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Temperature effect on chemical-induced carcinogenesis in hamster cheek pouch
Environmental Toxicology and Pharmacology 26 (2008) 147–149
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Temperature effect on chemical-induced carcinogenesis in hamster cheek pouch K. Kathiresan ∗ , N. Sithrangaboopathy Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, Tamilnadu 608502, India
a r t i c l e
i n f o
Article history: Received 6 December 2007 Received in revised form 5 February 2008 Accepted 3 March 2008 Available online 19 March 2008 Keywords: Temperature Oral cancer DMBA Hamster Antioxidant
a b s t r a c t 7,12-Dimethylbenz[a]anthracene (DMBA), a potent chemical carcinogen, was used to induce oral cancer on hamster buccal pouch, under two temperature regimes (22 ± 2 and 28 ± 2 ◦ C) for 25 weeks of observation. The animal group under high temperature showed rapid tumour incidence and weight loss. It also exhibited biochemical changes such as reduced lipid peroxidation in the oral tumour tissue, accompanied by significant increase in the levels of reduced glutathione, glutathione peroxidase and glutathione-stransferase. Therefore, we propose that elevated temperature is a cofactor, accelerating the process of DMBA-induced carcinogenesis in hamster cheek pouch. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Squamous cell carcinoma of the oral cavity is one of the most common cancers in the world and it is the third major cancer in India (Shanta et al., 1994). Intra-oral mucosa in humans may be exposed frequently to polyaromatic hydrocarbons from tobacco consumption, barbecued foods, other carcinogens and modifying agents such as ethanol, food additives, and radiations. However, rate of the cancer incidence may differ with environmental conditions. Temperature is one of the cofactors in human skin carcinogenesis. It is also experimentally proved that long-term exposure to increased temperature as encountered in skin areas exposed to excessive sunlight, i.e. in sunburn, leads to tumour development (Boukamp et al., 1999). Elevated temperature, as measured in the areas of sunburn, may contribute to skin cancer by generating DNA damage through oxidative stress pathway (Cerutti et al., 1990). The effect of temperature on oral cancer is largely unknown and hence the present study was made to find out its effect in chemically induced oral cancer on hamster cheek pouch. 2. Material and method
tion (NIN) Hyderabad, India. The animals were housed in polypropylene cages at a rate of 6 per cage and maintained at two temperature regimes: 22 ± 2 ◦ C (termed as group 1) and 28 ± 2 ◦ C (group 2) separately under 12-h light and dark cycle. All the animals were fed with a standard pellet diet (Mysore Snack Feed Ltd., Mysore) and water ad libitum. 2.2. Chemicals Dimethylbenz[a]anthracene (DMBA) was purchased from Sigma Chemical Company, USA and all other reagents used were of analytical grade. 2.3. Treatment schedule A total of 60 animals were used for the two temperature regimes. All the animals were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouch using a No. 4 brush, three times a week continuously for a period of up to 25 weeks. Each application left approximately 0.4 mg of DMBA per animal. The animals were analyzed for feed consumption and live weight at weekly intervals. They were also analyzed for number, volume and burden and histopathological changes at 5week intervals from 5th week of the experiment up to 25 weeks. At the regular interval of every 5 weeks, the animals were sacrificed and the buccal cavity tissues were dissected out carefully. The tumour volume was determined by measuring the amount of water displaced, when the dissected tumour tissue was introduced in a graduated beaker containing a known quantity of water. The fresh tissues were analyzed for enzymes and the preserved tissues in 10% formalin were analyzed for histopathology.
2.1. Animals 2.4. Biochemical analysis All the experiments were carried out with male Syrian hamsters, each weighing at 100 ± 10 g and aged for 9 ± 1 weeks, obtained from the National Institute of Nutri-
∗ Corresponding author. Tel.: +91 4144 243223. E-mail address: [email protected] (K. Kathiresan). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.03.007
Thiobarbituric acid reactive substances (TBARS) released from endogenous lipid peroxides reflecting the lipid peroxidation process were assayed in tissues as described by Ohkawa et al. (1979). The pink coloured chromogen formed by the reaction of 2-thiobarbituric acid with the break down products of lipid peroxidation was read at 535 nm. Reduced glutathione (GSH) was determined by the method of Ellman (1959) based on the development of a yellow colour when 5,5 -dithiobis
148
K. Kathiresan, N. Sithrangaboopathy / Environmental Toxicology and Pharmacology 26 (2008) 147–149 Table 3 Lipid peroxidation in terms of thiobabituric acid reactive substances (TBARS) in oral pouch mucosa of experimental animal groups (means ± S.D.; n = 6)
Table 1 Influence of temperature on feed consumption and body weight of the hamsters during carcinogenesis (means ± S.D.; n = 6) Weeks of observation 5 10 15 20 25
Feed consumption (g animal−1 )
Body weight (g animal−1 )
22 ± 2 ◦ Ca
22 ± 2 ◦ Cc
5.8 6.0 6.0 5.6 5.3
± ± ± ± ±
28 ± 2 ◦ Cb
0.62 0.70 0.68 0.58 0.50
6.45 6.65 5.80 3.10 1.50
± ± ± ± ±
0.65 0.72 0.68 0.40 0.20
106.5 113.1 117.8 115.5 105.0
± ± ± ± ±
Lipid peroxidationa
Weeks of analysis
22 ± 2 ◦ Ca
28 ± 2 ◦ Cd
10 12 13 12 10
107.5 112.2 109.2 85.0 75.0
± ± ± ± ±
5 10 15 20 25
10 11 10 08 05
80.58 75.26 67.43 59.54 54.28
± ± ± ± ±
28 ± 2 ◦ Cb
7.12 6.23 5.81 5.52 5.17
75.51 69.35 53.61 49.70 42.51
± ± ± ± ±
6.16 5.85 5.42 4.45 4.31
Feed consumption and body weight are significantly varied at p < 0.05 in different temperature regimes.
Values are significantly varied at p < 0.05 in different temperature regimes. a nmol/100 mg protein.
(2-nitrobenzoic acid) was added to compounds containing sulphydryl groups. Glutathione peroxidase (GPx) activity was assayed by the method of Rotruck et al. (1973). A known amount of enzyme preparation was incubated with hydrogen peroxide in the presence of reduced glutathione for a specified time period. The amount of hydrogen peroxide utilized was determined by estimating GSH content by the method of Ellman (1959).
perature (group 2) exhibited early incidence of tumour at 8th week onwards, whereas those under low temperature (group 1) showed delayed incidence of tumour at 15th week onwards. The mean number of tumours as well as tumour burden was significantly higher in group 2 than in 1. The mean tumour burden was 3.15 and 5.06 ml in the groups 1 and 2, respectively. The body weight increased up to 10 weeks of observation under both temperature regions; however, reduction in body weight observed from 15th week onwards. At that time, the weight loss was higher in elevated temperature than at low temperature and also significantly feed consumption was low.
2.5. Histopathological studies The tissues were embedded in paraffin and the resulting sections were stained with hematoxylin and eosin, and then were examined at magnification (10×) using a standard optical microscope. 2.6. Statistical analysis
3.2. Histological observation
One-way ANOVA and Duncan multiple range tests were used to compare mean values at 0.05 probabilities.
Table 2 summarizes the incidence of oral neoplasms and histopathological changes in two different temperature regimes. Well-developed squamous cell carcinomas with a number of epithelial and keratin pearls in the connective tissue with cellular pleomorphism were observed in group 2 (Fig. 1).
3. Results 3.1. Gross observations Table 1 summarizes the feed consumption of the experimental animals. A significant change in the feed consumption behaviour was observed in the later stage of the experiment, but not in the initial stages. Table 2 indicates the tumour incidence and tumour burden of the animal groups tested. The animals under high tem-
3.3. Biochemical observations Lipid peroxidation in the buccal pouch of experimental animals is given in Table 3. The lipid peroxidation level in hamsters under
Table 2 Tumour incidence and histopathological changes in experimental animals maintained under two temperature regimes (means ± S.D.; n = 6) Weeks of observation
Number of tumours ◦
5 10 15 20 25
Tumour volume (ml) ◦
◦
◦
Tumour burden (ml)a
Histo Pathological changes
22 ± 2 C
28 ± 2 C
22 ± 2 C
28 ± 2 C
22 ± 2 ◦ Cc
28 ± 2 ◦ Cd
22 ± 2 ◦ C
28 ± 2 ◦ C
– – – 5 7
– 5 7 9 11
– – – 0.32 ± 0.012 0.45 ± 0.025
– 0.28 ± 0.34 ± 0.40 ± 0.46 ±
– – – 1.60 ± 0.005 3.15 ± 0.012
– 1.4 ± 2.38 ± 3.6 ± 5.06 ±
– – ++ +++ +++
– ++ +++ +++ +++
a
b
0.011 0.012 0.025 0.030
0.003 0.010 0.013 0.42
Tumour volume and tumour burden are significantly varied at p < 0.05 in different temperature regimes. + hyperplasia, ++ dysphasia, +++ squamous cell carcinoma. a Tumour burden = number of tumours × volume of tumour.
Fig. 1. Histological changes in hamster cheek pouch tissue showing less keratin pearl formation in the animals maintained at 22 ± 2 ◦ C (A), and well-developed keratin pearls with cellular pleomorphism at 28 ± 2 ◦ C (B) on 15th week of observation. (Hematoxylin and eosin stain. Original magnification ×10.)
K. Kathiresan, N. Sithrangaboopathy / Environmental Toxicology and Pharmacology 26 (2008) 147–149
149
Table 4 Antioxidant status of experimental animals under two temperature regimes (means ± S.D.; n = 6) Weeks of analysis
◦
5 10 15 20 25
GPx (Ua /g protein)
GSH (mg/g tissue) ◦
28 ± 2 C
22 ± 2 C
28 ± 2 C
22 ± 2 ◦ Ce
28 ± 2 ◦ Cf
0.15 ± 0.010 0.25 ± 0.021 0.32 ± 0.023 0.39 ± 0.028 0.45 ± 0.032
0.23 ± 0.011 0.36 ± 0.028 0.45 ± 0.035 0.54 ± 0.042 0.63 ± 0.052
5.69 ± 0.48 6.75 ± 0.53 7.26 ± 0.58 8.55 ± 0.65 9.86 ± 0.67
6.64 ± 0.45 7.55 ± 0.52 9.10 ± 0.61 10.25 ± 0.76 14.45 ± 0.81
0.52 ± 0.042 0.85 ± 0.051 1.25 ± 0.072 1.64 ± 0.075 2.32 ± 0.081
0.65 ± 0.042 1.52 ± 0.065 1.75 ± 0.071 2.52 ± 0.082 3.65 ± 0.085
b
◦
GST (Ub /g protein)
22 ± 2 C
a
c
◦
d
GSH, GPx and GST are significantly varied at p < 0.05 in different temperature regimes. a mol glutathione utilized/min. b mol, 1-chloro-2,4-dinitrobenzene (CDNB)-reduced glutathione (GSH) conjugate formed/min.
elevated temperature (28 ± 2 ◦ C) was lower than that under low temperature (22 ± 2 ◦ C). Table 4 shows the activities of GSH, GPx and the levels of glutathione in the buccal pouch mucosa of two different experimental animal groups. In elevated temperature, the levels of GSH and the activities of GPx and GST were remarkably higher than those under low temperature. 4. Discussion It is clear from the data that high temperature accelerated the process of DMBA-induced carcinogenesis to a magnitude of 2.5 times on hamster cheek pouch (Table 2). Such of the stimulatory effect of temperature is not known till date for oral cancer. Temperature, as a result of sunburn or heat, is one of the major cofactors involving in the process of carcinogenesis and it is wellknown for skin cancer (Cerutti et al., 1990). Earlier in vitro studies reveal that genetic instability is a potential mechanism by which elevated temperature causes tumorigenic conversion. Temperature at 40 ◦ C is able to induce DNA strand breaks which eventually result in chromosomal aberrations (Boukamp et al., 1999). Rat embryo fibroblasts when cultured at 39 ◦ C as compared to 37 ◦ C exhibit an increased number of strand breaks and that these cells become tumorigenic when maintained at 39 ◦ C for more than 7 days (Marczynska et al., 1980). Also the high temperature decreases effectiveness of the repair of UV-induced damage to DNA (Goss and Parsons, 1976). The animals under elevated temperature showed an increased feed consumption and body weight at the earlier stage of the experiment up to 15 weeks of observation (Table 1). This may be due to accelerated metabolic activities in the animals under stress of elevated temperature. However, these animals showed gross weight loss after 15th week of observation due to less feed consumption as well as the tumour burden (Tables 1 and 2). Lipid peroxides are recognized to play an important role in the control of cell division. An inverse relationship has been observed between lipid peroxidation and the rate of cell proliferation; the tumours exhibit low levels of lipid peroxidation (Diplock et al., 1994). GSH plays a vital role in important cellular functions which include maintenance of thiol status of proteins, destruction of H2 O2 , translocation of amino acids across cell membranes, detoxification of foreign compounds and biotransformation of drugs (Mans et al., 1992). In the present study, decline in the lipid peroxidation in
DMBA-induced oral tumours was found associated with enhanced levels of GSH, GPx and GST (Tables 3 and 4). The GSH, which is the substrate for GPx and GST, has regulatory effects on cell proliferation. The GSH, GPx and GST are over-expressed in a wide variety of tumours (Diplock et al., 1994). The present study is consistent with the hypothesis of Slater et al. (1984) that a decrease in lipid peroxidation is associated with an increase in antioxidant enzymes, and this favours growth of tumour cells, as was found in the present study with animals under elevated temperature (Tables 1–3). Acknowledgements The authors are thankful to the authorities of Annamalai University for providing facilities. References Boukamp, P., Popp, S., Bleuel, K., Tomakidi, E., Burkle, A., Fusenig, N.E., 1999. Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature. Oncogene 18, 5638–5645. Cerutti, P., Amstad, P., Larsson, R., Shah, G., Krupitza, G., 1990. Mechanisms of oxidant carcinogenesis. Prog. Clin. Biol. Res. 347, 183–186. Diplock, A.T., Rice-Evans, A.C., Burton, R.H., 1994. Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention? Cancer Res. 54, 1952–1956. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Goss, P., Parsons, P.G., 1976. Temperature-sensitive DNA repair of ultraviolet damage in human cell lines. Int. J. Cancer 17, 296–303. Mans, D.R., Schurhuis, G.J., Treskes, M., Lafleur, M.V., Retel, J., Pinedo, Lankelma, J., 1992. Modulation by DL-butathione – S, R-sulphomixine of etoposide toxicity on human non-small cell lung, ovarian and breast carcinoma cell line. Br. J. Cancer 28A, 1447–1452. Marczynska, B., McPheron, L., Wilbanks, G.D., Tsurumoto, D.M., Deinhardt, F., 1980. Attempts to transform primate cells in vitro by herpes simplex virus. Exp. Cell Biol. 48, 114–125. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., Hoekstra, W.G., 1973. Selenium: biochemical roles as a component of glutathione peroxidase. Science 179, 588–590. Shanta, V., Gajalakshmi, C.K., Swaminathan, R., Ravichandran, K., Vasanti, L., 1994. Cancer registration in Madras metropolitan tumor registry India. Eur. J. Cancer 30, 974–978. Slater, T.F., Benedetto, C., Burton, G.W., Cheeseman, K.H., Ingold, K.U., Nodes, J.T., 1984. Lipid peroxidation in animal tumours. A disturbance in the control of cell division. In: Thaler, H., Crastes dePaulet, A., Paoletti, A. (Eds.), Eicosanoids and Cancer. Raven Press, New York, pp. 1–29.
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Temperature effect on chemical-induced carcinogenesis in hamster cheek pouch K. Kathiresan ∗ , N. Sithrangaboopathy Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, Tamilnadu 608502, India
a r t i c l e
i n f o
Article history: Received 6 December 2007 Received in revised form 5 February 2008 Accepted 3 March 2008 Available online 19 March 2008 Keywords: Temperature Oral cancer DMBA Hamster Antioxidant
a b s t r a c t 7,12-Dimethylbenz[a]anthracene (DMBA), a potent chemical carcinogen, was used to induce oral cancer on hamster buccal pouch, under two temperature regimes (22 ± 2 and 28 ± 2 ◦ C) for 25 weeks of observation. The animal group under high temperature showed rapid tumour incidence and weight loss. It also exhibited biochemical changes such as reduced lipid peroxidation in the oral tumour tissue, accompanied by significant increase in the levels of reduced glutathione, glutathione peroxidase and glutathione-stransferase. Therefore, we propose that elevated temperature is a cofactor, accelerating the process of DMBA-induced carcinogenesis in hamster cheek pouch. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Squamous cell carcinoma of the oral cavity is one of the most common cancers in the world and it is the third major cancer in India (Shanta et al., 1994). Intra-oral mucosa in humans may be exposed frequently to polyaromatic hydrocarbons from tobacco consumption, barbecued foods, other carcinogens and modifying agents such as ethanol, food additives, and radiations. However, rate of the cancer incidence may differ with environmental conditions. Temperature is one of the cofactors in human skin carcinogenesis. It is also experimentally proved that long-term exposure to increased temperature as encountered in skin areas exposed to excessive sunlight, i.e. in sunburn, leads to tumour development (Boukamp et al., 1999). Elevated temperature, as measured in the areas of sunburn, may contribute to skin cancer by generating DNA damage through oxidative stress pathway (Cerutti et al., 1990). The effect of temperature on oral cancer is largely unknown and hence the present study was made to find out its effect in chemically induced oral cancer on hamster cheek pouch. 2. Material and method
tion (NIN) Hyderabad, India. The animals were housed in polypropylene cages at a rate of 6 per cage and maintained at two temperature regimes: 22 ± 2 ◦ C (termed as group 1) and 28 ± 2 ◦ C (group 2) separately under 12-h light and dark cycle. All the animals were fed with a standard pellet diet (Mysore Snack Feed Ltd., Mysore) and water ad libitum. 2.2. Chemicals Dimethylbenz[a]anthracene (DMBA) was purchased from Sigma Chemical Company, USA and all other reagents used were of analytical grade. 2.3. Treatment schedule A total of 60 animals were used for the two temperature regimes. All the animals were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouch using a No. 4 brush, three times a week continuously for a period of up to 25 weeks. Each application left approximately 0.4 mg of DMBA per animal. The animals were analyzed for feed consumption and live weight at weekly intervals. They were also analyzed for number, volume and burden and histopathological changes at 5week intervals from 5th week of the experiment up to 25 weeks. At the regular interval of every 5 weeks, the animals were sacrificed and the buccal cavity tissues were dissected out carefully. The tumour volume was determined by measuring the amount of water displaced, when the dissected tumour tissue was introduced in a graduated beaker containing a known quantity of water. The fresh tissues were analyzed for enzymes and the preserved tissues in 10% formalin were analyzed for histopathology.
2.1. Animals 2.4. Biochemical analysis All the experiments were carried out with male Syrian hamsters, each weighing at 100 ± 10 g and aged for 9 ± 1 weeks, obtained from the National Institute of Nutri-
∗ Corresponding author. Tel.: +91 4144 243223. E-mail address: [email protected] (K. Kathiresan). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.03.007
Thiobarbituric acid reactive substances (TBARS) released from endogenous lipid peroxides reflecting the lipid peroxidation process were assayed in tissues as described by Ohkawa et al. (1979). The pink coloured chromogen formed by the reaction of 2-thiobarbituric acid with the break down products of lipid peroxidation was read at 535 nm. Reduced glutathione (GSH) was determined by the method of Ellman (1959) based on the development of a yellow colour when 5,5 -dithiobis
148
K. Kathiresan, N. Sithrangaboopathy / Environmental Toxicology and Pharmacology 26 (2008) 147–149 Table 3 Lipid peroxidation in terms of thiobabituric acid reactive substances (TBARS) in oral pouch mucosa of experimental animal groups (means ± S.D.; n = 6)
Table 1 Influence of temperature on feed consumption and body weight of the hamsters during carcinogenesis (means ± S.D.; n = 6) Weeks of observation 5 10 15 20 25
Feed consumption (g animal−1 )
Body weight (g animal−1 )
22 ± 2 ◦ Ca
22 ± 2 ◦ Cc
5.8 6.0 6.0 5.6 5.3
± ± ± ± ±
28 ± 2 ◦ Cb
0.62 0.70 0.68 0.58 0.50
6.45 6.65 5.80 3.10 1.50
± ± ± ± ±
0.65 0.72 0.68 0.40 0.20
106.5 113.1 117.8 115.5 105.0
± ± ± ± ±
Lipid peroxidationa
Weeks of analysis
22 ± 2 ◦ Ca
28 ± 2 ◦ Cd
10 12 13 12 10
107.5 112.2 109.2 85.0 75.0
± ± ± ± ±
5 10 15 20 25
10 11 10 08 05
80.58 75.26 67.43 59.54 54.28
± ± ± ± ±
28 ± 2 ◦ Cb
7.12 6.23 5.81 5.52 5.17
75.51 69.35 53.61 49.70 42.51
± ± ± ± ±
6.16 5.85 5.42 4.45 4.31
Feed consumption and body weight are significantly varied at p < 0.05 in different temperature regimes.
Values are significantly varied at p < 0.05 in different temperature regimes. a nmol/100 mg protein.
(2-nitrobenzoic acid) was added to compounds containing sulphydryl groups. Glutathione peroxidase (GPx) activity was assayed by the method of Rotruck et al. (1973). A known amount of enzyme preparation was incubated with hydrogen peroxide in the presence of reduced glutathione for a specified time period. The amount of hydrogen peroxide utilized was determined by estimating GSH content by the method of Ellman (1959).
perature (group 2) exhibited early incidence of tumour at 8th week onwards, whereas those under low temperature (group 1) showed delayed incidence of tumour at 15th week onwards. The mean number of tumours as well as tumour burden was significantly higher in group 2 than in 1. The mean tumour burden was 3.15 and 5.06 ml in the groups 1 and 2, respectively. The body weight increased up to 10 weeks of observation under both temperature regions; however, reduction in body weight observed from 15th week onwards. At that time, the weight loss was higher in elevated temperature than at low temperature and also significantly feed consumption was low.
2.5. Histopathological studies The tissues were embedded in paraffin and the resulting sections were stained with hematoxylin and eosin, and then were examined at magnification (10×) using a standard optical microscope. 2.6. Statistical analysis
3.2. Histological observation
One-way ANOVA and Duncan multiple range tests were used to compare mean values at 0.05 probabilities.
Table 2 summarizes the incidence of oral neoplasms and histopathological changes in two different temperature regimes. Well-developed squamous cell carcinomas with a number of epithelial and keratin pearls in the connective tissue with cellular pleomorphism were observed in group 2 (Fig. 1).
3. Results 3.1. Gross observations Table 1 summarizes the feed consumption of the experimental animals. A significant change in the feed consumption behaviour was observed in the later stage of the experiment, but not in the initial stages. Table 2 indicates the tumour incidence and tumour burden of the animal groups tested. The animals under high tem-
3.3. Biochemical observations Lipid peroxidation in the buccal pouch of experimental animals is given in Table 3. The lipid peroxidation level in hamsters under
Table 2 Tumour incidence and histopathological changes in experimental animals maintained under two temperature regimes (means ± S.D.; n = 6) Weeks of observation
Number of tumours ◦
5 10 15 20 25
Tumour volume (ml) ◦
◦
◦
Tumour burden (ml)a
Histo Pathological changes
22 ± 2 C
28 ± 2 C
22 ± 2 C
28 ± 2 C
22 ± 2 ◦ Cc
28 ± 2 ◦ Cd
22 ± 2 ◦ C
28 ± 2 ◦ C
– – – 5 7
– 5 7 9 11
– – – 0.32 ± 0.012 0.45 ± 0.025
– 0.28 ± 0.34 ± 0.40 ± 0.46 ±
– – – 1.60 ± 0.005 3.15 ± 0.012
– 1.4 ± 2.38 ± 3.6 ± 5.06 ±
– – ++ +++ +++
– ++ +++ +++ +++
a
b
0.011 0.012 0.025 0.030
0.003 0.010 0.013 0.42
Tumour volume and tumour burden are significantly varied at p < 0.05 in different temperature regimes. + hyperplasia, ++ dysphasia, +++ squamous cell carcinoma. a Tumour burden = number of tumours × volume of tumour.
Fig. 1. Histological changes in hamster cheek pouch tissue showing less keratin pearl formation in the animals maintained at 22 ± 2 ◦ C (A), and well-developed keratin pearls with cellular pleomorphism at 28 ± 2 ◦ C (B) on 15th week of observation. (Hematoxylin and eosin stain. Original magnification ×10.)
K. Kathiresan, N. Sithrangaboopathy / Environmental Toxicology and Pharmacology 26 (2008) 147–149
149
Table 4 Antioxidant status of experimental animals under two temperature regimes (means ± S.D.; n = 6) Weeks of analysis
◦
5 10 15 20 25
GPx (Ua /g protein)
GSH (mg/g tissue) ◦
28 ± 2 C
22 ± 2 C
28 ± 2 C
22 ± 2 ◦ Ce
28 ± 2 ◦ Cf
0.15 ± 0.010 0.25 ± 0.021 0.32 ± 0.023 0.39 ± 0.028 0.45 ± 0.032
0.23 ± 0.011 0.36 ± 0.028 0.45 ± 0.035 0.54 ± 0.042 0.63 ± 0.052
5.69 ± 0.48 6.75 ± 0.53 7.26 ± 0.58 8.55 ± 0.65 9.86 ± 0.67
6.64 ± 0.45 7.55 ± 0.52 9.10 ± 0.61 10.25 ± 0.76 14.45 ± 0.81
0.52 ± 0.042 0.85 ± 0.051 1.25 ± 0.072 1.64 ± 0.075 2.32 ± 0.081
0.65 ± 0.042 1.52 ± 0.065 1.75 ± 0.071 2.52 ± 0.082 3.65 ± 0.085
b
◦
GST (Ub /g protein)
22 ± 2 C
a
c
◦
d
GSH, GPx and GST are significantly varied at p < 0.05 in different temperature regimes. a mol glutathione utilized/min. b mol, 1-chloro-2,4-dinitrobenzene (CDNB)-reduced glutathione (GSH) conjugate formed/min.
elevated temperature (28 ± 2 ◦ C) was lower than that under low temperature (22 ± 2 ◦ C). Table 4 shows the activities of GSH, GPx and the levels of glutathione in the buccal pouch mucosa of two different experimental animal groups. In elevated temperature, the levels of GSH and the activities of GPx and GST were remarkably higher than those under low temperature. 4. Discussion It is clear from the data that high temperature accelerated the process of DMBA-induced carcinogenesis to a magnitude of 2.5 times on hamster cheek pouch (Table 2). Such of the stimulatory effect of temperature is not known till date for oral cancer. Temperature, as a result of sunburn or heat, is one of the major cofactors involving in the process of carcinogenesis and it is wellknown for skin cancer (Cerutti et al., 1990). Earlier in vitro studies reveal that genetic instability is a potential mechanism by which elevated temperature causes tumorigenic conversion. Temperature at 40 ◦ C is able to induce DNA strand breaks which eventually result in chromosomal aberrations (Boukamp et al., 1999). Rat embryo fibroblasts when cultured at 39 ◦ C as compared to 37 ◦ C exhibit an increased number of strand breaks and that these cells become tumorigenic when maintained at 39 ◦ C for more than 7 days (Marczynska et al., 1980). Also the high temperature decreases effectiveness of the repair of UV-induced damage to DNA (Goss and Parsons, 1976). The animals under elevated temperature showed an increased feed consumption and body weight at the earlier stage of the experiment up to 15 weeks of observation (Table 1). This may be due to accelerated metabolic activities in the animals under stress of elevated temperature. However, these animals showed gross weight loss after 15th week of observation due to less feed consumption as well as the tumour burden (Tables 1 and 2). Lipid peroxides are recognized to play an important role in the control of cell division. An inverse relationship has been observed between lipid peroxidation and the rate of cell proliferation; the tumours exhibit low levels of lipid peroxidation (Diplock et al., 1994). GSH plays a vital role in important cellular functions which include maintenance of thiol status of proteins, destruction of H2 O2 , translocation of amino acids across cell membranes, detoxification of foreign compounds and biotransformation of drugs (Mans et al., 1992). In the present study, decline in the lipid peroxidation in
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