Zinc and Zinc Related Enzymes in Precancerous and Cancerous Tissue in the Colon of Dimethyl Hydrazine Treated Rats

Cancer of the large bowel has been the subject of intensive investigations for many years and its incidence has been found to vary with geographic area and socioeconomic level (Fabricant & Broitman, 1990). Colorectal cancer is one of the most common malignancies in industrialized countries and one of the most common causes of cancerrelated death. From the distribution of large bowel cancer in various parts of the world and data dealing with migrant populations, it is apparent that environmental factors, rather than genetic and social factors, play a significant role in the etiology of colon cancer in man (Haenszel et al., 1973). The trace element zinc has been demonstrated to be essential for the growth, development and differentiation of all types of life, including microorganisms, plants and animals (Vallee, 1986). Zinc plays a major role in protein synthesis and has an important function in gene expression; the involvement in gene expression is both structural and an enzymatic role. These proteins require zinc for their conformation and DNA binding abilities (Berg, 1990; Choo et al., 1997). The zinc atoms are an integral, firmly bound part of the proteins molecule and are often involved in the active site. They also contribute to the conformation


Introduction
Cancer of the large bowel has been the subject of intensive investigations for many years and its incidence has been found to vary with geographic area and socioeconomic level (Fabricant & Broitman, 1990).Colorectal cancer is one of the most common malignancies in industrialized countries and one of the most common causes of cancer-related death.From the distribution of large bowel cancer in various parts of the world and data dealing with migrant populations, it is apparent that environmental factors, rather than genetic and social factors, play a significant role in the etiology of colon cancer in man (Haenszel et al., 1973).
The trace element zinc has been demonstrated to be essential for the growth, development and differentiation of all types of life, including microorganisms, plants and animals (Vallee, 1986).Zinc plays a major role in protein synthesis and has an important function in gene expression; the involvement in gene expression is both structural and an enzymatic role.These proteins require zinc for their conformation and DNA binding abilities (Berg, 1990;Choo et al., 1997).The zinc atoms are an integral, firmly bound part of the proteins molecule and are often involved in the active site.They also contribute to the conformation

Zinc and Zinc Related Enzymes in Precancerous and Cancerous Tissue in the Colon of Dimethyl Hydrazine Treated Rats
Pamela Christudoss 1* , R Selvakumar 1 , Anna B Pulimood 2 , Jude Joseph Fleming 1 , George Mathew 3 and structural stability of many metalloenzymes.Decrease in zinc metalloenzymes activity along with decrease in zinc varies with different zinc enzymes and is dependent on the tissue, enzyme turnover rate, and affinity of the enzyme for zinc.(Rhodes & Klug, 1993) The plasma and tissue zinc levels in various diseases have been the subject of a multitude of investigations and possible involvement of zinc has been recognized in many cancerous conditions.The role of zinc in carcinoma development has been the subject of debate and reports of zinc values in the biological fluids from cancer patients are often conflicting and contradictory.Hence the mechanism by which plasma zinc and tissue zinc decrease in various cancerous tissues is still obscure (Ehud et al., 1983).
Colorectal cancer, which is a common cause of cancer related deaths, is often due to persistent oxidative stress leading to DNA damage.Oxidative stress caused by reactive oxygen species, (ROS) results in damage to cellular structure, and this has frequently been implicated in the initiation and promotion phases of carcinogenesis.Levels of ROS are controlled by the antioxidant defense system.Several components of this system are micronutrients (e.g.vitamins C and E) or enzymes produced by specific endogenous pathways.(e.g.CuZn and Mn superoxide dismutase) (Evans & Halliwell, 2001 ;Vertuani et al., 2004).Super oxide dismutases (SOD) are metalloenzymes that play a vital role in the protection of aerobic cells against oxygen toxicity (Cindy & Yi Feng, 1999).Tissue CuZnSOD activity is reduced in a number of malignancies.Some tumors have less CuZn SOD in comparison with the more metabolically active tissues (Grigolo et al., 1998).
The E.coli alkaline phosphatase (ALP) was first recognized as a zinc metalloenzyme in 1962.It is a marker enzyme of microvillus membranes (MVM) in the intestine and exists both in the soluble and membrane bound forms (Katica et al., 2006).Intestinal ALP has been shown to decrease in patients with gastric or colonic cancer (Sotaro et al., 2006).Studies in tissue homogenates indicates that the activity of alkaline phosphatase in tissues decrease markedly when a zinc deficient state is induced in experimental animals as compared with pair fed controls (Reinhold & Kfoury, 1969).
Development of colon cancer is a multistep process involving a series of pathological alterations ranging from discrete microscopic lesions like aberrant crypt foci (ACF), also known as preneoplastic lesions to malignant tumors (Bird, 1995).It is believed that colon cancer, like the majority of cancers, evolves from precursor lesions also known as adenomatous polyps (Elizabeth, 1988;Farber & Cameron, 1980).To identify the possible precursor lesions of adenomatous polyps of colon cancer, several investigators have studied the development of colon cancer in rodents treated with chemical carcinogens.Carcinogens are administered to experimental animals like rats and mice, to study the effect of certain diets and preventive agents on tumor induction (Day & Morson, 1978;Richards, 1977).
The genotoxic chemical 1, 2-DMH (symmetrical dimethyl hydrazine) is one of the agents most frequently used in experimental models of colon carcinogenesis.It is a complete carcinogen that induces the initiation and promotion steps of carcinogenesis yielding macroscopically visible neoplasms (Park 1997;Sequeira, 2000).Regardless of the mode of administration, DMH specifically induces tumors within the descending colon and the histopathology is similar to that observed for human sporadic colon tumors (Rogers & Nauss, 1985).Numerous studies have been carried out to investigate the protective effect of various chemicals, drugs and food items on the induction and development of DMH induced colon tumors in rats, (Anisimov & Popovich, 1997;Cameron et al., 1997;Furkawa, 1997) but so far, to the best of our knowledge no study has shown the relationship between changes in tissue zinc levels and zinc related enzymes in DMH induced colon carcinogenesis.Thus the DMH induced colon cancer model in rats is a good tool to investigate the relationship of zinc and zinc related enzymes to colon cancer.In a study reported by Davis et al, [1994] using the DMH model for induction of carcinoma, tumors were found after the administration of DMH at a dosage of 30mg/kg twice a week for 5 months.Hence we used a similar dose of 30mg/kg DMH twice a week for 3 months and sacrificed rats at the fourth month in order to induce the development of precancerous lesions in the colon.Since we found an association between tissue zinc levels and zinc related enzymes such as SOD and alkaline phosphatase in the colon in precancerous stage we decided to further set up a colon carcinoma model by injecting rats with DMH (30mg/kg body wt.) twice a week for 4 months and sacrificing them at 6 months.
The aim of this study was to investigate whether the biochemical changes in the colon with respect to tissue zinc levels and zinc related enzymes such as CuZnSOD and Alkaline phosphatase are associated with the histological changes that take place in the colonic mucosa from normal to precancerous stage and then to the cancerous stage.

Materials and Methods
Animals: Thirty two adult Wistar rats (100-120g) at six weeks of age, obtained from the Institutional animal house were housed in polypropylene plastic cages, in an animal holding room under controlled conditions with 25 ± 2º C, 50 ± 10% humidity, and 12 hour light -dark cycles.The rats were allowed water and food ad libitum, observed daily and weighed weekly.This study was approved by the Animal Experimentation Ethics Committee of our Institution.

Experimental design and induction of colonic tumors.
Thirty two rats were randomly assigned to two groups, Group A (Precancerous) and Group B (cancerous).All groups were fed the same diet and maintained as described.Group A was further subdivided into control group (n=6) and experimental group (n=8), which received subcutaneous dose of saline or 30mg/kg body weight DMH dissolved in saline respectively twice a week for 3 months and were euthanized at 4 months.
Group B was further subdivided into control group (n=6) and experimental group (n=12), which received a subcutaneous dose of saline or 30mg/kg body weight DMH dissolved in saline respectively twice a week for 4 months and were euthanized at 6 months.All groups were euthanized by chloroform inhalation.Blood was collected at the time of sacrifice for estimation for plasma zinc.
Tissue preparation-histopathology, measurement of tissue zinc, CuZnSOD, Alkaline phosphatase, plasma zinc: all rats were examined grossly at necropsy.The entire intestine from the stomach to anus was removed and the large intestine was isolated.It was slit length wise, washed in saline and the mucosal surface examined for gross pathology.Any lesions detected were measured, location noted and dissected.A portion was taken for DOI:http://dx.doi.org/10.7314/APJCP.2012.13.2.487 Zinc and Zinc Related Enzymes in the Colon of DMH Treated Rats histological examination after fixation in 10% w/v formaldehyde overnight and was routinely processed as per standard methods, embedded in paraffin, sectioned at 5µm, stained with hematoxylin and eosin for evaluation by light microscopy.At autopsy, a section of the colon of the rats of both the groups A and B and a section of the stomach , small intestine and liver of the rats of group B were harvested, washed with ice cold saline and stored at -20 º C until analysis for tissue zinc estimation as described (Kahnke ,1966]. The mucosa of the stomach, small intestine, and large intestine were used for cytosolic homogenate preparation using phosphate buffered saline pH 7.4.CuZnSOD activity (units/mg/protein) in the homogenate was measured by MTT reduction as described [Kim,2000) Activity of alkaline phosphatase (µmoll/min/mg protein) was assayed as described (Dorai ,1977).
Plasma zinc was estimated as described (Rosner & Garfien, 1968).Zinc determination was carried out using Perkin Elmer AAS model-100.The concentration of tissue zinc is reported in µg/g dry weight tissue, with an appropriate blank being used for correction of zinc content in the acids used for digestion.
Ethanediol-stabilised QC serum, prepared in our laboratory, was run along with each batch of plasma zinc estimations.The small intestine from a control rat was cut into many pieces, and processed for tissue zinc with every batch of samples.Similarly the mucosa of the small intestine and large intestine of control rat was aliquoted into storage tubes and included with each batch for the estimation of CuZnSOD and Alkaline phosphatase activity respectively.

Statistical Analysis
Data are expressed as Mean ± SD.Differences between groups were analyzed using Non-Parametric test -Mann Whitney U test and Kruskal Wallis test.A difference was considered statistically significant when the probability associated with it was less than 0.05 (p<0.05).

A) DMH induced precancer model
In the DMH induced precancerous model, colonic precancerous stage was established at 4 months in animals that were treated with DMH.The colon of control group that received vehicle saline was grossly normal.Precancerous stage of the colon was observed upon histological examination in 87.5% animals (7 out of 8) that were treated with DMH and sacrificed after 4 months.Histological examination of the large intestine of rats, showed proliferated glands lined by cells with hyper chromatic pseudo stratified nuclei and cytoplasmic -mucin depletion consistent with dysplasia (Figure 1).
Plasma zinc levels in the DMH treated rats at four months showed a significant decrease (mean 23%) as compared with the control group (93 ±9 vs 121±4.5µg/dl, p<0.01).As shown in Fig 2 (A), the mean tissue zinc levels (wet weight) in the large intestine showed a significant decrease as compared with controls (mean  46%, (p<0.005), the dry weight analysis also showed a similar decrease (Figure 2B).CuZnSOD activity in the mucosal homogenate of the large intestine showed a significant decrease (mean 24%, p<0.05), as compared with controls (Figure 2C).The alkaline phosphatase activity in the mucosal homogenate in the large intestine was significantly lower (mean 27.3%, p<0.05) as compared with controls (Figure 2D).

B) DMH induced cancerous model
In the DMH induced carcinoma model, the colon of the control group of rats that received saline was grossly The mean plasma zinc levels in the DMH induced carcinoma model was significantly lower (mean 33.4%, p<0.01) as compared with control group treated with saline.(81±11 vs. 122±4.3µg/dl).As shown in Figure 5A, tissue zinc levels (wet weight) in the large intestine showed a significant decrease as compared with the control group (mean 72%, p<0.001).Dry weight analysis showed a similar % mean decrease in tissue zinc levels (Fig 5B).CuZnSOD activity in the mucosal homogenate of the large intestine shows a mean 45% decrease, which was statistically significant as compared with controls (p<0.001),(Fig 5C).A significant decrease in alkaline phosphatase activity in the mucosal homogenate of the large intestine was observed (51% mean) as compared with controls (p<0.001)(Fig 5D).
In the small intestine and in the body of the stomach, a significant decrease in the tissue zinc levels (dry weight, mean 44%, p<0.01, and mean 42%, p<0.01 respectively) was observed as compared with controls (Fig 5B).As shown in Fig 5C, the CuZnSOD activity in the small intestine showed a significant decrease (mean 19%, p<0.05) as compared with controls, where as in the stomach the CuZn SOD activity remained unaltered.The Alkaline phosphatase activity in the stomach and in the small intestine remained unaltered (Fig 5D).The liver showed a significant decrease in tissue zinc levels (P<0.05,mean 34%) as compared with controls.(Figure 5 A, B).CuZnSOD and alkaline phosphatase activity were not measured in the liver.Histological examination of the liver showed scattered hepatocytes, mild nuclear enlargement and hyperchromasia (Figure 6).

Quality control
Quality control (QC) values for tissue zinc, plasma zinc and tissue SOD are presented below.QC values for plasma zinc showed a CV of 2.7%, for tissue zinc a CV of 3.75%, for CuZnSOD activity and alkaline phosphatase activity a CV of 1.2 % and 3.6 % respectively.

Discussion
Zinc plays an important role in cellular physiology and this element functions as a specific activator of many enzymatic reactions (Hambidge, 1989).Zinc deficiency is implicated in the development and progression in some cancers (Valcovic, 1980).Initiation of mucosal changes, which progress to preneoplastic lesions, may be necessary in the triggering of the adenoma-carcinoma sequence (Sandforth, 1988).The present study, in DMH induced colon precancerous and cancerous stage in the rat model, examined the relationship between plasma zinc, colonic tissue zinc levels and the zinc requiring enzymes CuZnSOD and Alkaline phosphatase activity in the progression from normal to precancerous stage and then to the cancerous stage.
In the DMH induced precancerous model, histologically severe dysplasia was observed in the colon of majority of rats (87.5%) at 4 months.Early precancerous lesions established in the proximal and distal regions of the   normal and showed normal morphology (Figure 4A) Out of the 12 experimental rats that received DMH for four months and sacrificed at 6 months, 11 rats (91.6%) showed tumors in the large intestine.Macroscopical examination of the resected colon of the 11 rats showed tumors varying between diameters of 2mm to 1.5 cm (Fig. 3).Out of the 11 rats which showed macroscopically large tumors, 4 rats (30%) showed tumors in the ascending colon and the remaining 7 rats (60%) showed tumors in the descending colon.Three of these rats showed multiple tumors in the colon (2 to 4 small size tumors).Colon carcinoma was colon was associated with a significant decrease in the concentration of plasma zinc, colonic tissue zinc and zinc requiring enzymes CuZnSOD and alkaline phosphatase in the DMH treated rats at 4 months as compared with controls.In the DMH induced colon carcinoma model, the results show that colon carcinoma was histologically established in the majority of rats (92%) at 6 months.In agreement with other studies we found maximum tumors in the distal colon (Corpet & Tache, 2002).Histologically differentiated DMH induced colon cancer was directly related to a greater significant decrease in the concentration of plasma zinc, colonic tissue zinc and zinc related SOD and alkaline phosphatase activities as compared to DMH induced precancerous model.The trend towards a gradual decrease in zinc concentration in the malignant tissue in our study is in agreement with the results reported earlier in cancer patients with respect to the differences in normal -malignant tissue zinc (Sadamitsu & Sadao, 1978;Brown,1980).The exact cause of low zinc levels in the malignant tissue is not known.Neoplastic diseases have been reported to lead to changes in zinc metabolism as evidenced by alterations in plasma zinc concentration (Rao, Ganesh, 1998).Mc bean et al suggested that the diseased conditioned may lead to an overall zinc decrease in the tissues (Mc Bean, 1972).
It has been documented that significant oxidative stress, caused by free radicals, occurs in carcinoma of the intestinal mucosa.In particular, the super oxide radical ion has been postulated as the possible cause of cancer (Oberley, Buttner, 1979).As observed in previous studies, altered activities of superoxide dismutase were shown to be important in multistage carcinogenesis of both rodents and humans.They showed that tumors possessed decreased SOD activity compared to metabolically active tissues (Van Driel, 1997;Dionisi,1975;Westman & Stefan, 1981) The results in our study illustrates that CuZnSOD activity is impaired in the colonic precancerous tissue at four months and in the colonic cancerous tissue at six months of DMH treatment.This may mean that it might have a functional role in human colon carcinogenesis.The decreased concentration of the trace element zinc found in the precancerous tissue and cancerous tissue may contribute to the differences observed in tissue CuZnSOD enzyme activities.
Intestinal enzymes associated with the striated cell border, such as intestinal alkaline phosphatase may be useful markers for malignant expression in colonocytes (Brown et al., 1980).The results in this study are consistent with the findings of studies reported earlier, in that a decrease in alkaline Phosphatase was observed in tumors (Harmenberg,1991) indicating that these enzymes are very sensitive to zinc depletion.The decrease in alkaline phoshatase in the cancer tissue was greater than the decrease in the precancer tissue.These studies indicate modifications in the enzyme structure that could be brought about by zinc decrease (Reinhold & Kfoury, 1969).Intestinal alkaline phosphatase was found to decrease in gastric or colonic cancer with an exacerbation of its cancerous lesions as compared to higher activity in normal individuals (Katsuyuki, 1978).
Studies have shown that tumors develop in various regions of the gastrointestinal tract and small bowel when DMH was administered to rats (Zhurkov, 1996).The present study, in the DMH treated carcinoma model a significant decrease in tissue zinc levels and CuZnSOD activity was observed in various parts of the gastrointestinal tract i.e. body of stomach, and in small intestine.Trans mural changes along the wall of the gastrointestinal tract could be the cause of this change.These findings led to the hypothesis that early mucosal alterations, which are preneoplastic lesions, and which can lead on to further development of carcinogenesis might also lead to a decrease in tissue zinc concentration and zinc-requiring enzyme CuZnSOD and alkaline phosphatase activity in the large intestine and to possible changes in other parts of the gastrointestinal tract.
In this study, we also observed significant decrease in tissue zinc levels in the liver in DMH treated rats at six months.Mild histological changes leading to atypical hyperplasia and nuclear enlargement were observed.As reported in earlier studies, the presence of atypical cells in the liver suggests that besides gastrointestinal tumors, the liver may also be a site for abnormalities (Zhurkov, 1996;Inagake,1995).The significant decrease in liver zinc in our studies could be attributed to the histological changes taking place with administration of DMH, which occurs alongside with the progression of dysplasia in the colon.
The above findings support the view that decreasing tissue zinc levels and zinc requiring enzymes SOD and alkaline phosphatase are representative of the biochemical alterations in the colonic mucosa, which may in turn reflect the cellular changes that progressed to dysplasia and frank malignancy.In our study, histologically preneoplastic lesions in the large intestine also presented progressive changes.At fourth month, in the DMH treated group, the colon exhibited hyperplastic changes, while frank malignancy was established at 6 months of DMH treatment.These may be sequentially related to the biochemical events in the large intestine in DMH treated rats.In this study, the decreased concentration of the trace element zinc found in the precancerous colon tissue and a greater decrease observed in the cancerous colonic tissue may contribute to the decreases observed in tissue CuZnSOD and alkaline phosphatase enzyme activities in the colon in the precancerous and the cancerous models.
In conclusion, this study indicates that there is a decrease in plasma zinc, tissue zinc concentration, CuZnSOD activity, and alkaline phosphatase activity which occurs alongside the changes in the colonic mucosa during precancerous and cancerous transformation.It may be finally suggested that the zinc decrease in tissue is associated with carcinogenesis which may limit the functions of zinc requiring enzymes such as CuZnSOD and alkaline phosphatase.

Figure 1 .
Figure 1.Light Microscopy of Large Intestine of DMH Treated Rats at 4 Months Showing Precancerous Stage, Colonic Mucosa with Closely Packed Glands Lined by Epithelial Cells Exhibiting Dysplasia.Magnification 40 xs

Figure 3 .
Figure 3. Large Intestine of Rat Showing Large Tumor Growth in the Colon (Colon of Rat Injected with DMH at 6 months)

Figure 5 .
Figure 5. Zinc Status in Various O rgans.(A) wet wt, (B) dry wt.; C) CuZnSOD (D) Alkaline phosphatase activity in large intestine, small intestine and stomach (body) in DMH treated rats as compared to controls-at 6 months.Values represent mean ± SD from 6 rats, * p<0.05, ** p<0.001 when compared to control.

Figure 6 .
Figure 6.Light Microscopy of Liver.A) normal liver tissue of rat treated with saline, B) Liver of DMH treated rat at 6 months showing liver with scattered hepatocytes, mild nuclear enlargement and hyperchromasia.

Figure 4 .
Figure 4. Light Microscopy of the Colon in DMH Treated Rats at 6 Months.A) Normal colonic tissue, magnification 40 xs, B) Colonic mucosa with infiltrating adenocarcinoma composed of irregular glands lined by malignant columnar epithelial cells infiltrating the stroma.Magnification 40 xs