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Experimental Biology and Medicine 228:689-696 (2003)
© 2003 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Zinc Accumulation in N-Methyl-N-Nitrosourea-Induced Rat Mammary Tumors Is Accompanied by an Altered Expression of ZnT-1 and Metallothionein

R. Lee, W. Woo, B. Wu, A. Kummer, H. Duminy and Z. Xu1

Food, Nutrition, and Health Program, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Zinc is essential for cell proliferation. Several human studies have shown that in breast cancer tissues, zinc concentration expressed on a per tissue weight basis is higher than that in normal breast tissues. However, the mechanisms involved are unknown. N-methyl-N-nitrosourea (MNU)-induced rat mammary tumorigenesis is one of the most widely used rodent mammary tumorigenesis models for studying human breast cancer due to their similarities in hormone dependency, pathogenesis, histological classification, and immunocytochemical markers. This study was to establish if there was an accumulation of zinc in MNU-induced rat mammary tumors and, if there was, to explore the possible mechanisms involved. Sprague-Dawley rats were sham-treated or MNU-treated (50 mg/kg; n = 12) for 100 days. In MNU-induced mammary tumors (mammary tumors), zinc concentration expressed on a per dry weight basis was 12 times of that in normal mammary glands. Moreover, the mRNA level of ZnT-1 (a transporter involved in zinc efflux) in mammary tumors was reduced by 55% as compared with that in normal mammary glands. The mRNA level of Nramp2 (a divalent cation importer) and ZnT-4 (another transporter involved in zinc efflux) was unaffected by MNU-induced mammary tumorigenesis. The mRNA and protein levels of metallothionein (a putative zinc storage protein) in mammary tumors were 1.3 and 3.5 times of that in normal mammary glands, respectively. Collectively, our observations showed that zinc is accumulated in MNU-induced rat mammary tumors and this accumulation is accompanied by an altered expression of ZnT-1 and metallothionein, suggesting that zinc homeostasis might be altered in MNU-induced rat mammary tumorigenesis. Because zinc is essential to cell proliferation and cell proliferation is increased in mammary tumors, zinc accumulation is likely a part of an integrated effort to ensure sufficient zinc supply to sustain tumor growth.

Key Words: metallothionein • mammary tumorogenesis • Nramp2 • ZnT-1 • ZnT-4 • zinc


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Zinc has been shown to be important to tumor growth in several animal model systems. For example, zinc depletion suppresses the growth of implanted mammary carcinoma (1), Lewis lung tumors (2), leukemia (3), Walker 256 carcinoma (4, 5), and several types of solid tumors (3). Furthermore, zinc depletion increases the survival rate of animals implanted with Lewis lung tumors (2) or with Walker 256 carcinoma (4, 5). Evidently, zinc adequacy influences the outcome of tumorigenesis in these animal model systems.

Some human studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues when it is expressed on the basis of either wet tissue weight (6–8) or dry tissue weight (6, 9–11). Causation of this elevated zinc concentration in breast cancer tissues and its significance in breast cancer development are presently unknown. Normal breast tissues contain a considerable amount of fatty tissues, which are low in mineral content, including zinc (12). In contrast, breast cancer tissues are comprised of tightly packed cancer cells (13) and zinc in the body is primarily associated with proteins (14). Therefore, one of the possible causes of an elevated zinc concentration in breast cancer tissues simply reflects the histological and biochemical differences between breast cancer and normal breast tissues.

Another possible cause is that the elevated zinc concentration results from an altered zinc homeostasis in breast cancer tissues. Cellular zinc concentration is homeostatically regulated. Although the mechanisms involved are not well understood, zinc homeostatic regulation is achieved at the level of influx, efflux, and retention. Zinc influx and efflux involves transporters. Natural resistance-associated macrophage protein 2 (Nramp2; also known as divalent cation transporter 1 [DCT1] and divalent metal transporter 1 [DMT1]) is a member of the Nramp family of transporters. Nramp2 is a divalent cation transporter involved in influx of a broad range of metals, including Zn2+ (15). Besides Nramp2, there are two families of zinc transporters: the ZIP (Zrt, Irt-like proteins) family and the CDF (cation diffusion facilitator) family (16). ZIP family transporters have prominent roles in zinc influx. However, the ZIP family transporters in mammals have presently been reported only in humans and mouse, but not yet in rats. The CDF family transporters are involved in the efflux of zinc, including both from the cytoplasm out of the cell and into organellar compartments. Presently, five members of the CDF family have been identified in mammals: ZnT-1 (17), ZnT-2 (18), ZnT-3 (19), ZnT-4 (20), and ZnT-5 (21). Upon entering the cells, excess zinc is bound to metallothionein, which has been suggested to function as a zinc storage protein (22). If cellular zinc concentration is viewed as the net balance among zinc influx, efflux, and retention, then an altered expression of these transporters and metallothionein could result in altered cellular and thus tissue zinc concentrations. Because zinc is essential for cell proliferation (23–25) and considering its demonstrated importance in tumor growth (1–5), the elevated zinc concentration in breast cancer tissues could be an indication of its involvement in breast cancer development. Therefore, it becomes important to elucidate the possible causes of the elevated zinc concentration in breast cancer tissues.

N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis in rats is one of the most widely used rodent mammary tumorigenesis models for studying human breast cancer (26–28). MNU-induced rat mammary tumorigenesis and human breast cancer development share many similarities in hormone dependency, pathogenesis, histological classification, and immunocytochemical markers. Due to these similarities, MNU-induced rat mammary tumorigenesis is a useful experimental model for the study of human breast cancer development. However, the involvement of zinc in MNU-induced rat mammary tumorigenesis presently is not clear.

We hypothesized that zinc is accumulated in MNU-induced rat mammary tumorigenesis through an altered zinc homeostasis. The objective of this study was to establish if zinc was accumulated in MNU-induced rat mammary tumors as seen in human breast cancer tissues and, if there was, to determine the possible mechanisms involved. The results reported herein showed an increased zinc concentration in MNU-induced rat mammary tumors as compared with that in normal mammary glands. In mammary tumors, ZnT-1 expression was suppressed, whereas metallothionein expression was upregulated. Because zinc is essential to cell proliferation and tumorigenesis is characterized by uncontrolled cell proliferation, these observations collectively suggest that zinc accumulation in mammary tumors is likely a part of an integrated effort to ensure a sufficient zinc supply to sustain the growth of MNU-induced rat mammary tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Mammary Tumorigenesis.
Twenty-one-day-old, female Sprague-Dawley rats (Animal Care Center, University of British Columbia, British Columbia, Canada) were either sham-treated (saline containing 0.05% acetic acid; pH 4) or MNU-treated (50 mg/kg body weight; n = 12 rats/group; using the procedures described earlier) (29). The rats were cared for according to the guidelines of the Canadian Council on Animal Care. One hundred days after MNU injection, mammary glands (normal mammary glands) were removed from the sham-treated rats. From the tumor-bearing rats, both MNU-induced mammary tumors (mammary tumors) and mammary glands (tumor-free mammary glands) were removed. Fatty tissues and mammary gland tissues were carefully trimmed from the tumors. The tumor-free mammary glands removed from the tumor-bearing rats were carefully inspected to ensure that there was no presence of visible tumor.

Tissue Composition and Tissue Zinc Concentrations.
To determine zinc concentration, normal mammary glands, tumor-free mammary glands, and mammary tumors (~1 g/sample) were dried in acid-washed crucibles overnight at 100°C to a constant weight. The dried tissues were then transferred quantitatively to acid-washed glass test tubes and were wet-ashed with 3 mL of concentrated nitric acid at 70°C overnight (30). Each test tube was covered with an acid-washed glass marble to prevent evaporation. Upon completion of digestion, the wet-ashed tissue samples were transferred quantitatively to 5- or 10-mL acid-washed volumetric flasks and brought to volume with double-deionized water. The digested tissue samples were further diluted with 0.1 N nitric acid to an appropriate concentration for the determination of tissue zinc concentration using flame atomic absorption spectrophotometer (Atomic Absorption Spectrophotometer, model 2380; Perkin Elmer, Norwalk, CT). The tissue zinc concentration was normalized on a per dry tissue weight or on a per protein basis. The tissue moisture content was calculated from the drying process described above. The tissue protein concentration was determined using the Lowry’s method (31). The tissue lipid concentration was determined using the Folch’s method (32).

Metallothionein Protein.
Metallothionein protein level in normal mammary glands, tumor-free mammary glands, and mammary tumors was assessed using the cadmium (109Cd)-hemoglobin affinity assay (33). The sensitivity of this assay can be improved by reducing the concentration of Cd solution used while the assay reproducibility can be improved by reducing the background counts (34). Accordingly, we made three modifications: first, we reduced the concentration of Cd solution to 0.4 µg Cd/ml; second, we repeated hemoglobin addition three times; and third, we increased the centrifugation speed to 14,000g. 109Cd radioactivity was counted using a gamma counter (1277 Gammamaster; LKB Wallac, Turku, Finland).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Total tissue RNAs were isolated using the single-step RNA isolation procedure (35). Briefly, tissues (200 mg) were homogenized in denaturing solution (2 mL) with a glass-Teflon tissue grinder. The homogenate was then mixed with sodium acetate (2 M; 0.2 mL), water-saturated phenol (2 mL), and chloroform:isoamyl alcohol (49:1; 0.4 mL). After incubation (for 15 min at 4°C) and centrifugation (9,500 rpm for 20 min at 4°C), total RNA was precipitated with 100% isopropanol at -20°C. RNA pellets were resuspended in DEPC-treated water and stored at -70°C until analysis. RT-PCR was performed using the ThermoScript RT-PCR System plus Platinum Taq DNA Polymerase (Invitrogen, Burlington, Ontario, Canada) according to the manufacturer’s instruction. Total RNA (2 µg) was used for reverse transcription. The resulting cDNA (2 µl, which is equivalent to 0.2 µg of total RNA.) was subsequently amplified with PCR (GeneAmp PCR System 2400; Perkin Elmer). The PCR conditions used were adopted from the conditions described earlier (36) with modifications to optimize the amplification. Briefly, after a hot start (94°C/2 min), the samples were amplified at 94°C for 20 sec, 55°C for 30 sec, and 72°C for 40 sec followed by a final extension at 72°C for 5 min. For each target gene, preliminary experiments were conducted to determine the relationship between amplification efficiency and the number of cycles. Based on these preliminary experimental results, the number of cycles used was chosen to ensure several things. First, the amplification was within the liner phase of the amplification curve. The R2 for the number of cycles tested for Nramp2, ZnT-1, ZnT-4, metallothionein, and ß-actin was 0.97, 0.97, 0.99, 0.92, and 0.99, respectively. Second, the amplification did not yield nonspecific PCR product. In general, a higher number of cycles was used for low abundant genes (i.e., Nramp2, ZnT-1, and ZnT-4), whereas a lower number of cycles was used for higher abundant genes (i.e., metallothionein and ß-actin). Specifically, the number of PCR cycles for Nramp2, ZnT-1, ZnT-4, metallothionein, and ß-actin was 35, 34, 36, 31, and 27, respectively. The sequences for the sense and antisense primers used are listed in Table IGo. Total RNAs isolated from the kidney, liver, and brain of untreated adult rats using the same isolation protocol described above were used as positive control for Nramp2, ZnT-1 and metallothionein, and ZnT-4, respectively. Two negative controls, cDNA blank and primer blank, were also included for each PCR amplification. ß-Actin was used as the control to verify the integrity of the RNA and equal loading. After the quantification of the optical density of the band using Scion Image (Release Beta 4.02; Scion Co., Frederick, MD), the level of Nramp2, ZnT-1, ZnT-4, and metallothionein mRNAs was normalized on the optical density of the corresponding ß-actin band.


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  		Table I. PCR Primers Used for Amplification of cDNAs
 
Statistical Analysis.
The differences among the means of the treatment groups were analyzed by one-way analysis of variance (ANOVA) and, where applicable, followed by Tukey’s Honest test (The SAS System for Windows Release 6.12; SAS Institute, Cary, NC). Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue Composition.
Composition of normal mammary glands, tumor-free mammary glands, and mammary tumors was assessed by determining moisture content, and protein and lipid concentrations. Tissue composition between normal mammary glands and tumor-free mammary glands were very similar (Table IIGo). Moisture content in mammary tumors was approximately three times of that in normal mammary glands and in tumor-free mammary glands. Protein concentration in mammary tumors was approximately eight and six times of that in normal mammary glands and in tumor-free mammary glands, respectively. Lipid concentration in mammary tumors was approximately one-half of that in normal mammary glands and in tumor-free mammary glands.


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  		Table II. Tissue Composition
 
Tissue Zinc Concentrations.
On a per dry weight basis, zinc concentration in mammary tumors was twelve and nine times of that in normal mammary glands and in tumor-free mammary glands, respectively (Fig. 1AGo). On a per protein basis, zinc concentration in mammary tumors was 1.4 times of that in normal mammary glands, but was the same as that in tumor-free mammary glands (Fig. 1BGo). Zinc concentration in normal mammary glands was the same as that in tumor-free mammary glands on either a per dry tissue or a per protein basis.



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  		Figure 1. Zinc concentration in normal mammary glands, tumor-free mammary glands, and mammary tumors. (A) Zinc concentration normalized on a per dry weight basis. (B) Zinc concentration normalized on a per protein basis. Values represent mean ± SEM (n = 12 rats). The means sharing a common letter are not significantly different (P < 0.05). Normal, normal mammary glands removed from sham-treated rats; Tumor-free, tumor-free mammary glands removed from tumor-bearing rats; Tumor, MNU-induced mammary tumors removed from tumor-bearing rats.

 
Nramp2, ZnT-1, and ZnT-4 mRNA Levels.
To elucidate the possible causes of elevated zinc concentration in mammary tumors, the expression of Nramp 2 and zinc transporters was assessed. Because zinc concentration was the same between the normal and tumor-free mammary glands expressed on a per dry weight and a per protein bases, Nramp 2 and zinc transporter expressions were only assessed in normal mammary glands and mammary tumors. Nramp2 mRNA level in mammary tumors was the same as that in normal mammary glands (Fig. 2Go). Compared with normal mammary glands, ZnT-1 mRNA level in mammary tumors was reduced by 55% (Fig. 3Go). ZnT-4 mRNA level in mammary tumors was also the same as that in normal mammary glands (Fig. 4Go).



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  		Figure 2. RT-PCR analysis of Nramp2 mRNA level in normal mammary glands and mammary tumors. Total RNAs were isolated from normal mammary glands and mammary tumors were reverse transcribed as described in "Materials and Methods." PCR products were subjected to agarose (1%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative Nramp2 mRNA levels. Lane 1, DNA ladder; Lane 2, positive control (kidney); Lane 3, negative control (no cDNA); Lane 4, negative control (no primers); Lanes 5 and 6, normal mammary glands; Lanes 7 and 8, mammary tumors. (B) Relative Nramp2 mRNA level. Values represent mean ± SEM (n = 4). The means sharing a common letter are not significantly different (P < 0.05). (C) Ethidium bromide-stained agarose gel showing representative ß-actin mRNA levels. Lane 1, DNA ladder; Lane 2, negative control (no cDNA); Lane 3, negative control (no primers); Lane 4, liver; Lane 5, kidney; Lane 6, brain; Lanes 7 and 8, normal mammary glands; Lanes 9 and 10, mammary tumors. Control, positive controls. Normal, normal mammary glands from sham-treated rats; Tumor, MNU-induced mammary tumors from tumor-bearing rats.

 


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  		Figure 3. RT-PCR analysis of ZnT-1 mRNA level in normal mammary glands and mammary tumors. Total RNAs were isolated from normal mammary glands and mammary tumors were reverse transcribed as described in "Materials and Methods." PCR products were subjected to agarose (1%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative ZnT-1 mRNA levels. Lane 1, DNA ladder; Lane 2, positive control (liver); Lane 3, negative control (no cDNA); Lane 4, negative control (no primers); Lanes 5 and 6, normal mammary glands; Lanes 7 and 8, mammary tumors. (B) Relative ZnT-1 mRNA level. Values represent mean ± SEM (n = 4). The means sharing a common letter are not significantly different (P < 0.05). Normal, normal mammary glands removed from sham-treated rats; Tumor, MNU-induced mammary tumors removed from tumor-bearing rats.

 


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  		Figure 4. RT-PCR analysis of ZnT-4 mRNA level in normal mammary glands and mammary tumors. Total RNAs isolated from normal mammary glands and mammary tumors were reverse transcribed as described in "Materials and Methods." PCR products were subjected to agarose (1%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative ZnT-4 mRNA levels. Lane 1, DNA ladder; Lane 2, positive control (kidney); Lane 3, negative control (no cDNA); Lane 4, negative control (no primers); Lanes 5 and 6, normal mammary glands; Lanes 7 and 8, mammary tumors. (B) Relative ZnT-4 mRNA level. Values represent mean ± SEM (n = 4). The means sharing a common letter are not significantly different (P < 0.05). Normal, normal mammary glands removed from sham-treated rats; Tumor, MNU-induced mammary tumors removed from tumor-bearing rats.

 
Metallothionein mRNA and Protein Levels.
Metallothionein is considered a zinc storage protein. Metallothionein protein level in mammary tumors was three and four times of that in normal mammary glands and in tumor-free mammary glands, respectively (Fig. 5Go). Because metallothionein level was the same between normal and tumor-free mammary glands, we then focused on metallothionein mRNA level in mammary tumors and normal mammary glands. In mammary tumors, metallothionein mRNA level was 1.3 times of that in normal mammary glands (Fig. 6Go).



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  		Figure 5. Metallothionein protein level in normal mammary glands, tumor-free mammary glands, and mammary tumors. Values represent mean ± SEM (n = 5 rats, except in mammary tumors, n = 6 rats). The means sharing a common superscript are not significantly different (P < 0.05). Normal, normal mammary glands removed from sham-treated rats; Tumor-free, tumor-free mammary glands removed from MNU-treated rats; Tumor, MNU-induced mammary tumors removed from tumor-bearing rats.

 


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  		Figure 6. RT-PCR analysis of metallothionein mRNA level in normal mammary glands and mammary tumors. Total RNAs isolated from normal mammary glands and mammary tumors were reverse transcribed as described in "Materials and Methods." PCR products were subjected to agarose (1%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative metallothionein mRNA levels. Lane 1, DNA ladder; Lane 2, positive control (kidney); Lane 3, negative control (no cDNA); Lane 4, negative control (no primers); Lanes 5 and 6, normal mammary glands; Lanes 7 and 8, mammary tumors. (B) Relative metallothionein mRNA level. Values represent mean ± SEM (n = 4). The means sharing a common letter are not significantly different (P < 0.05). MT, metallothionein; Normal, normal mammary glands removed from sham-treated rats; Tumor, MNU-induced mammary tumors removed from tumor-bearing rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have observed an elevated zinc concentration in mammary tumors. Cellular and thus tissue zinc concentrations are homeostatically regulated. Alteration to an otherwise balanced influx, efflux, and cellular retention would ultimately affect tissue zinc concentrations. In mammary tumors, Nramp2 expression remained at the same level as in normal mammary glands, but ZnT-1 expression was decreased by 55% as compared with that in normal mammary glands, suggesting that the elevated zinc concentration in MNU-induced mammary tumors is likely the result of reduced zinc efflux.

The elevated zinc concentration in mammary tumors coincided with upregulated metallothionein expression and increased metallothionein protein level. One of the established biological functions of metallothionein is maintaining zinc homeostasis through formation of the metallothionein-zinc complex (38). In this capacity, metallothionein functions as a zinc storage protein (22). Tissue metallothionein levels are closely related to tissue zinc concentration (39). Injection of zinc or feeding high-zinc diets increases tissue metallothionein levels such that most of the additional tissue zinc generally is bound to metallothionein (40, 41). Furthermore, it is known that free ionic zinc in biological systems is homeostatically maintained at very low concentrations. Thus, it appears that metallothionein represents a zinc accumulation in mammary tumors. It also is important to note that the amount of zinc accumulated exceeded the binding capacity of the additional complement of metallothionein protein, suggesting that the zinc accumulated in the mammary tumor is likely associated with proteins other than metallothionein. Because information about zinc and mammary tumorigenesis is limited, the nature of other zinc-binding species in the abnormal tissue remains to be defined. Together with the receptor expression data, zinc accumulation in mammary tumors appears to be a result of an altered zinc homeostasis characterized by a reduced ZnT-1 expression and an upregulated metallothionein expression. Further studies are needed to firmly establish the involvement of ZnT-1 and metallothionein in zinc accumulation in mammary tumors.

The significance of zinc accumulation in mammary tumors is not clear. However, zinc is essential to cell proliferation and is required for DNA and RNA syntheses. In cultured cells, zinc deficiency inhibits DNA synthesis (24, 42) and causes G1/S arrest in the cell cycle (23, 24). Zinc repletion results in increased DNA synthesis and cell proliferation (24). Similarly, dietary zinc deficiency in rats results in a decline of DNA synthesis (43). To sustain a higher rate of cell proliferation, such as in the case of tumorigenesis, there would need to be an increase in zinc. It is then perceivable that the machinery involved in zinc homeostasis would be geared up to meet such demand. Thus, accumulation of zinc in mammary tumors, along with a suppressed ZnT-1 expression and an upregulated metallothionein expression, could be an indication of the importance of zinc in MNU-induced rat mammary tumorigenesis.

Metallothionein mRNA and protein levels were elevated in MNU-induced rat mammary tumors. This observation is supported by observations in human breast cancer (44–47). In humans, overexpression of metallothionein in breast cancer tissues is primarily associated with more aggressive and higher grade tumors (46–49) and with poorer prognosis (44, 48–50). For example, metallothionein 1F (46) and 2A (47) mRNA and protein levels are higher in grade 3 tumors than in grade 1 and 2 tumors (46, 47). Although the precise role of metallothionein in the development of human breast cancer has yet to be established, several lines of evidence suggest that metallothionein plays a role in the proliferation of breast cancer cells. In human breast cancer tissues, metallothionein expression is significantly correlated to the Ki-67 index (47), which is considered as a reliable and reproducible marker for cell proliferative activity (51). Moreover, metallothionein is preferentially expressed in S-phase breast adenocarcinoma cells (52). In contrast, downregulation of metallothionein expression using antisense phosphorothioate oligomer inhibits cell growth and induces apoptosis (53). Therefore, upregulation of metallothionein expression in MNU-induced rat mammary tumors might be indicative of an increased cell proliferation.

Philcox and co-workers (54) reported a lack of relationship between metallothionein protein level and tumor progression in a transplanted mammary adenocarcinoma model. In that study, metallothionein protein level was only determined in tumors with various sizes (54). In contrast, upregulation of metallothionein reported herein was demonstrated by comparing metallothionein mRNA (Fig. 6Go) and protein (Fig. 5Go) levels in normal mammary glands with their levels in MNU-induced rat mammary tumors. Similarly, overexpression of metallothionein in human breast cancer is also shown by comparing metallothionein mRNA (46, 47) and protein (45–47) levels in normal breast tissues with their levels in breast cancerous tissues. The apparent inconsistency between Philcox’s study and the results reported herein could be due to the difference in experimental approach.

When normalized on a per dry weight basis, the zinc concentration in mammary tumors was twelve times of that in normal mammary glands and nine times of that in tumor-free mammary glands. However, when it was normalized on a per protein basis, the magnitude of zinc accumulation in mammary tumors was reduced to 1.4 times of that in normal mammary glands. Moreover, zinc concentration in mammary tumors was no longer higher than that in tumor-free mammary glands. This apparent contradiction can be attributed to the compositional differences among these tissues and the biochemical nature of zinc. In rats, mammary glands grow into the surrounding fatty tissues forming an inseparable mammary fat pad (26). In contrast, mammary tumors are composed of tightly packed tumor cells (13). This histological difference is reflected in the compositional difference where mammary tumors were high in protein, whereas mammary glands were high in fat as reported herein. In biological systems, zinc is mainly associated with proteins to exert their biological functions (e.g., zinc metalloenzymes and zinc-finger proteins) or to be bound to metallothionein. Therefore, it is more meaningful and accurate to compare zinc concentration on a per protein basis when its concentrations in different types of tissues, especially those tissues greatly differing in their protein and fat contents, are compared.

Results obtained from this study clearly demonstrate the accumulation of zinc in mammary tumors when compared with that in normal mammary glands. These results are consistent with previous observations in humans showing increased zinc concentration in cancerous breast tissues (9–11). Furthermore, the upregulation of metallothionein expression in rat mammary tumors was also consistent with the observations in human breast cancer tissues (44–47). These similarities between MNU-induced rat mammary tumors and human breast cancer tissues added more commonalities, making it a useful animal model for studying the role of trace metals such as zinc and metallothionein in human breast cancer.

It is interesting to note that even though both ZnT-1 and ZnT-4 are transporters involved in zinc efflux, ZnT-1 expression in mammary tumors was reduced, whereas ZnT-4 expression was unaffected. The relative abundance of ZnT-1 and ZnT-4 in tissues, including mammary glands and mammary tumors, is not known as well as the mechanisms involved in the regulation of their expressions. However, one possibility is that these two transporters are regulated by different mechanisms. For example, ZnT-1 expression in rat intestine is markedly upregulated when a supplemental zinc intake is provided, but ZnT-4 expression was refractory to changes in zinc intake (55). In contrast, ZnT-4 expression in mammary glands is upregulated during lactation, but ZnT-1 expression is unaffected (55). These observations suggest that expression of these two zinc transporters are regulated by different mechanisms. Further studies are needed to elucidate the mechanisms involved in the regulation of ZnT-1 and ZnT-4 gene expression in MNU-induced rat mammary tumorigenesis.

In summary, we have clearly shown the accumulation of zinc in MNU-induced rat mammary tumors. The zinc accumulation was accompanied by decreased ZnT-1 expression coupled with increased metallothionein expression. Significance of this zinc accumulation in MNU-induced rat mammary tumorigenesis requires further investigation. However, because zinc is essential to cell proliferation and tumorigenesis is characterized by uncontrolled cell proliferation, these observations collectively suggest that zinc accumulation in mammary tumors is likely a part of an integrated effort to ensure a sufficient zinc supply to sustain the growth of MNU-induced rat mammary tumors.


   Acknowledgments
 
The authors wish to thank Madeline Simpson and Bonnie Woo for their excellent technical assistance.


   Footnotes
 
This study was partially supported by an operating grant from Cancer Research, Inc. (to Z.X.).

1 To whom requests for reprints should be addressed at Food, Nutrition, and Health Program, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. E-mail: zxu{at}interchange.ubc.ca Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication July 15, 2002. Accepted for publication December 23, 2002.




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