- Email: [email protected]
Effect of mild whole body hyperthermia on cytotoxic action of N-methyl-N′-nitro-N-nitrosoguanidine in Swiss mice
Toxicology Letters 121 (2001) 63 – 68 www.elsevier.com/locate/toxlet
Effect of mild whole body hyperthermia on cytotoxic action of N-methyl-N%-nitro-N-nitrosoguanidine in Swiss mice Raghavendra S. Bagewadikar *, Manohar S. Patil, Asifa K. Zaidi, Mylvaganan Subramanian, Gangadhar S. Kaklij Radiation Biology Di6ision, Bhabha Atomic Research Centre, Mumbai 400085, India Received 22 August 2000; received in revised form 22 January 2001; accepted 22 January 2001
Abstract Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a modifier of N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) cytotoxicity in mice. Adult Swiss male mice, 6 – 8 weeks old, weighing about 25 g were exposed to mild WBH (39°C, 1 h) in a precision temperature controlled environmental chamber maintained at 50–60% relative humidity. Twenty-four hours after treatment, animals were administered with different doses of MNNG either by intraperitoneal (i.p.) injections or by feeding through drinking water and were monitored for survival. The studies revealed that the exposure of animals to mild WBH, 24 h prior to MNNG administration results in an increase in survival and recovery in mean body weight compared with those administered with MNNG only. This suggests that prior WBH treatment can effectively reduce the MNNG cytotoxicity in mice. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Whole body hyperthermia; MNNG; Cytotoxicity
1. Introduction In recent years much attention has been focused on protecting living organisms from harmful effects of radiations and from toxic effects of certain environmental and chemotherapeutic agents. Recent studies from our laboratory have shown that the exposure of mice to mild whole body hyperthermia (WBH) at 40°C for 1, 20 or 48 h prior to total body irradiation (TBI) with lethal * Corresponding author. Fax: 91225519613. E-mail address: [email protected] (R.S. Bagewadikar).
dose of g-rays afford significant protection as assessed by survival (Patil et al., 1996). Similar WBH induced radioprotection has been reported by Shen et al. (1991). WBH has also been shown to enhance the antitumour activity of chemotherapeutic agents such as carboplatin, and tumour necrosis factor (TNF) without toxic effects on normal tissues (Sakaguchi et al., 1994). This implies that mild WBH may also protect against toxicity of certain chemicals. N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) is known to mimic the effects of radiation such as induction of mutations (Mandel and
0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 3 1 6 - 2
64
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Greenberg, 1960; Adelberg et al., 1965), cancer (Schoental, 1996), chromosomal aberrations (Gichner et al., 1963) and cell death. It is an alkylating direct acting N-nitroso carcinogen and its mechanism of action is well studied in our laboratory (Bhattacharya and Bagewadikar, 1986; Bagewadikar and Bhattacharya, 1992). It is also highly toxic and shown to have growth inhibitory action on tumour cells (Aujard and Trincal, 1983). In view of this, it was considered of interest to ascertain whether mild WBH pre treatment will modulate MNNG induced cytotoxicity in mice. In the present communication, we report for the first time that mild WBH treatment when given 24 h prior to MNNG administration can effectively reduce MNNG toxicity in mice.
2. Materials and methods
2.1. Animals Syngenic Swiss mice, 6 – 8 weeks old, weighing approximately 25 g, maintained on stock laboratory diet and water ad libitum, were used. The use of animals was approved by the Institutional Animal Ethics Committee (IAEC) and the guidelines for the care of the animals were strictly followed throughout these studies.
2.3. Treatment of animals with MNNG In the first set of experiments MNNG (Fluka A.G. Switzerland) was administered through drinking water (1 mg/ml) to groups of mice (10 mice per group) for 1 and 2 weeks, 24 h after exposure to mild WBH. The control mice without WBH treatment were also fed same concentration of MNNG through drinking water. MNNG solution was supplied fresh every alternate day. Consumption of MNNG in drinking water was measured daily. After 1 and 2 weeks of MNNG treatment, the animals were monitored for their survival. In the second set of experiments, untreated and WBH treated mice received different concentrations of MNNG in saline (80, 120, 160 mg/kg body weight) by repeated i.p. injections 24 h after WBH treatment and were monitored for survival. The weight of each surviving animal was recorded periodically. The dead animals were subjected to postmortem and abdominal and thoracic viscera were examined grossly for deformities and solid tumours in organs.
2.4. Statistical analysis Results are expressed as mean9standard deviation (S.D.). Statistical significance was determined by use of the Log Rank test for survival.
2.2. Whole body hyperthermia treatment 3. Results Just before exposure to WBH, mice were injected intraperitoneally (i.p.) 1 ml of physiological saline (25°C) to compensate the water loss during hyperthermic treatment. For WBH treatment, unanaesthetised mice in groups of 10 were exposed to 39°C and relative humidity of 50 – 60% for 1 h in precision temperature controlled environmental chamber. The rectal temperature before, during and after WBH treatment was measured by a thermocouple probe. The body temperature raised to 40°C within 10 min of exposure and remained unchanged till the period of treatment. Temperature came down to a normal body temperature within 10 min after treatment.
The amount of MNNG consumed per group (10 mice per group) was calculated from daily water intake. Mice in WBH treated group consumed more water compared with untreated group. The amount of MNNG received after 1 week of feeding by untreated and WBH treated group was 86 and 123 mg, respectively. The results depicted in Fig. 1 show the effect of mild WBH on survival of mice against MNNG toxicity. It is evident from the figure that 90% of untreated mice fed with MNNG via drinking water (1 mg/ml for 1 week) died within 36 weeks. However, 50% of the mice exposed to mild WBH, 24 h prior to MNNG feeding survived for more
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
than 52 weeks even after receiving a high dose (43% higher dose) compared with WBH untreated group. Incidentally there was no difference in survival pattern between untreated and WBH treated animals when MNNG was fed for 2 weeks (results not shown). These results indicate that prior WBH treatment results in considerable reduction in MNNG induced toxicity in mice. Postmortem analysis of animals showed enlargement of spleen (Fig. 2a), liver, lymphnodes in most of the cases and solid tumours of lung (Fig. 2b) and small intestine (Fig. 2c) in animals that survived more than 40 weeks from MNNG treated group. This was not evident in WBH pre-treated group indicating that mild WBH protects animals from MNNG induced toxicity when given 24 h prior to MNNG administration. Since the feeding experiments were found to be extremely time consuming and also the dose of MNNG received by each animal was not uniform, the second set of experiments was carried out by i.p. administration of MNNG to mice. Results in Fig. 3 show the dose response of i.p. administered MNNG on survival of mice. It can be seen from the figure that MNNG was highly toxic at the doses of 120 –160 mg/kg body weight. None of the animals survived for more than 10 days after
Fig. 1. Effect of whole body hyperthermia on survival of mice against MNNG toxicity. Mice were given MNNG via drinking water (1 mg/ml, for 1 week) 24 h after WBH and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group. Statistical analysis, MNNG versus MNNG 24 h after WBH P B 0.1.
65
MNNG administration. However, 40% animals survived for more than 120 days at the dose of 80 mg/kg body weight. The next set of experiments on effect of WBH on MNNG toxicity was, therefore, carried out using 80 mg/kg body weight as a test dose. Results in Fig. 4 indicate that when MNNG was administered immediately before or after WBH, the survival of animals was decreased significantly. However, the animals which were given WBH 24 h prior to MNNG administration showed significant increase in survival compared with those who received MNNG only. This is corroborated by the recovery in mean body weight in WBH treated animals compared with those administered with MNNG only (Fig. 5). The data strongly suggest that prior WBH treatment results in protection against MNNG toxicity in mice.
4. Discussion The most interesting finding on WBH induced radioprotection (Shen et al., 1991; Patil et al., 1996) in mice prompted us to investigate whether prior WBH will also afford protection against toxic effects of radiomimetic compounds like MNNG. Our results reported in this communication serve to show that mild WBH (39°C, 1 h) treatment given 24 h prior to MNNG administration can afford significant protection in terms of survival of mice against MNNG cytotoxicity. The protective effect depends upon the time of MNNG administration after WBH treatment. The WBH induced increase in survival after MNNG administration corresponds very well with the recovery in mean body weight of WBH treated animals compared with those who received MNNG only. Furthermore, WBH induced reduction in MNNG cytotoxicity is confirmed by two independent modes of MNNG administration. The molecular mechanisms underlying the WBH induced reduction in MNNG toxicity are far from clear. Several recent studies have shown that heat shock proteins (HSPs) can protect cells from wide variety of stresses such as oxidant
66
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Fig. 2. Postmortem analysis of mice treated with MNNG: showing (a) enlarged spleen (b) solid tumor of lung (c) solid tumor of small intestine, from mice killed at 40 weeks after MNNG feeding via drinking water (1 mg/ml for 1 week). Small spleen in (a) is from mice treated with WBH + MNNG. No tumours were found in WBH+MNNG treated mice.
radicals or endotoxins (Hartl, 1996; Kiang and Tsokos, 1998). Frossard (1999) has reported excellent correlation between increased level of WBH induced HSP 70 expression in rat pancreas and increase in time to death in rats exposed to hyperthermia, when control and hyperthermia treated animals were killed using rapid asphyxia method with 100% CO2. Induction of HSPs in animals by WBH protects the brain and heart from tissue injury induced by ischemia (Liu et al., 1992; Brown and Sharp, 1999; Carroll and Yellon, 1999; Ohtsuka and Hata, 2000). Similarly, increased expression of HSPs results in extended life span in nematoda and the fruit fly (Smith, 1958; Lithgow et al., 1995; Tatar et al., 1997). It has also been reported that administration of cytokines such as transforming growth factor-b (TGF-b) exhibit myloprotection during chemotherapy with 5-flurouracil and cyclophosphamide (Lemoli et al., 1992; Carlino and Gregory, 1993; Grzegorzewski et al., 1994; Kekow et al., 1995). WBH treatment is known to increase
levels of various cytokines including TGF-b (Robins et al., 1995; Kekow et al., 1995). Kekow et al. (1995) have reported that transient increase
Fig. 3. Dose response of intraperitoneally administered MNNG on survival of mice. Mice were exposed to WBH (39°C for 1 h) 24 h prior to MNNG administration at 80 – 160 mg/kg bodyweight and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group.
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Fig. 4. Survival of mice treated with MNNG after WBH treatment. Mice were intraperitoneally administered with MNNG (80 mg/kg body weight), immediately before, immediately after and 24 h after WBH (39°C for 1 h) and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group. Statistical analysis, MNNG versus MNNG immediately before WBH P B 0.1; MNNG versus MNNG immediately after WBH PB 0.001; MNNG versus MNNG 24 h after WBH P B 0.05.
in TGF-b1 and TGF-b2 under WBH might play a role in protecting bone marrow from toxic effects of myelosuppressive chemotherapy. Thus, from the voluminous information available on WBH effects in biological systems, it would seem that induction of HSPs and certain cytokines after WBH might play an important role in protection against toxic effects of MNNG. This aspect is being currently investigated in our laboratory. In conclusion, our results seem to suggest that appropriate scheduling of WBH treatment and MNNG administration results in significant reduction in toxicity of even a highly potent genotoxic and proteotoxic chemical like MNNG. This implies that WBH may have a great potential in protecting against toxic effects of drug treatments and also in reducing the toxicity of chemotherapeutic agents used in cancer chemotherapy.
Acknowledgements We are thankful to V.G. Khankal and K.S. Gosavi for their excellent technical assistance.
67
Fig. 5. Effect of WBH on mean body weight of mice administered with MNNG (80 mg/kg body weight). The data represent the mean of three independent experiments.
References Adelberg, E.A., Mandel, M., Chen, G.C.C., 1965. Optimal conditions for mutagenesis by MNNG in E. Coli K12. Biochem. Biophys. Res. Commun. 18, 788– 795. Aujard, C., Trincal, G., 1983. The effects of N-methyl-N%-nitro-N-nitrosoguanidine on KB cells. 1.Growth inhibition and cell killing in relation to inhibition of macromolecular synthesis. Chem. Biol. Interact. 44, 79 – 93. Bagewadikar, R.S., Bhattacharya, R.K., 1992. Effect of Nmethyl-N%-nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach. J. Biochem. Toxicol. 7, 19 – 24. Bhattacharya, R.K., Bagewadikar, R.S., 1986. Functional modification of rat liver ribosomes by the in vitro action of N-methyl-N%-nitro-N-nitrosoguanidine. Chem. Biol. Interact. 57, 235– 251. Brown, I.R., Sharp, F.R., 1999. The cellular stress response in brain. In: Latchman, D.S. (Ed.), Stress Proteins. Springer, Berlin, pp. 243– 263. Carlino, J.A., Gregory, R.W., 1993. TGF-b2 decreases chemotherapy induced mortality in mice. J. Cell Biochem. 17, 61. Carroll, R., Yellon, D.M., 1999. Heat stress proteins and their relationship to myocardial protection. In: Latchman, D.S. (Ed.), Stress Proteins. Springer, Berlin, pp. 265– 279.
68
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Frossard, J.L., 1999. Heat shock protein 70 (HSP 70) prolongs survival in rats exposed to hyperthermia. Eur. J. Clin. Invest. 29, 561– 562. Gichner, T., Michaelis, A., Rieger, R., 1963. Radiomimetic effects of 1-methyl-3-nitro-1-nitrosoguanidine in Vicia faba. Biochem. Biophys. Res. Commun. 11, 120–124. Grzegorzewski, K., Ruscetti, F.W., Usui, N., et al., 1994. Recombinant transforming growth factor b1 and b2 protect mice from acutely lethal doses of 5-fluorouracil and doxorubicin. J. Exp. Med. 180, 1047–1057. Hartl, F.U., 1996. Molecular chaperones in cellular protein folding. Nature 381, 571–579. Kekow, J., Wiedemann, G.J., Katschinski, D.M., Kisro, J., Geisler, J., Wagner, T., Szymkowiak, C., 1995. Whole body hyperthermia increases plasma levels of transforming growth factor b. Int. J. Oncol. 7, 1427–1432. Kiang, J.G., Tsokos, G.C., 1998. Heat shock protein 70 kDa: molecular biology, biochemistry, physiology. Pharmacol. Ther. 80, 183– 201. Lemoli, R.M., Strife, A., Clarkson, B.D., et al., 1992. TGF-b3 protects normal human hemopoietic progenitor cells treated with 4-hydroperoxy cyclophosphamide in vitro. Exp. Hematol. 20, 12 – 52. Lithgow, G.S., White, T.M., Melov, S., Johnson, T.E., 1995. Thermotolerance and extended life span conferred by single gene mutations and induced by thermal stress. Proc. Natl. Acad. Sci. USA 92, 7540–7544. Liu, X., Engelman, R.M., Moraru, I.I., Rousou, J.A., Flack, J.E., Deaton, D.W., Moulik, N., Das, D.K., 1992. Heat shock: a new approach for myocardial preservation in cardiac surgery. Circulation 86, 11358–11363.
.
Mandel, J.D., Greenberg, J., 1960. A new chemical mutagen for bacteria, 1-methyl-3-nitro-1-nitrosoguanidine. Biochem. Biophys. Res. Commun. 3, 575– 577. Ohtsuka, K., Hata, M., 2000. Molecular chaperone function of mammalian HSP 70 and HSP 40: a review. Int. J. Hyperthermia 16, 231– 245. Patil, M.S., Kaklij, G.S., Poduval, T.B., Singh, B.B., 1996. Radioprotective effect of whole body hyperthermia on mice exposed to lethal doses of total body gamma irradiation. Ind. J. Exp. Biol. 34, 842– 844. Robins, H.I., Kutz, M., Wiedemann, G.J., Katschinski, D.M., Paul, D., Grosen, E., Tiggelaar, C.L., Spriggs, D., Gillis, W., d’Oleire, F., 1995. Cytokine induction by 41.8°C whole body hyperthermia. Cancer lett. 97, 195– 201. Sakaguchi, Y., Makino, M., Kaneko, T., Stephens, L.C., Strebel, F.R., Danhauser, L.L., Jenkins, G.N., Bull, J.M.C., 1994. Therapeutic efficacy of long duration-low temperature whole body hyperthermia when combined with tumor necrosis factor and carboplatin in rats. Cancer Res. 54, 2223– 2227. Schoental, R., 1996. Carcinogenic activity of N-methyl-N%-nitro-N-nitrosoguaninine. Nature 209, 726– 727. Shen, R.N., Hornback, N.B., Shidnia, H., Wu, B., Lu, L., Broxeyer, H.E., 1991. Whole body hyperthermia: a potent radioprotector in vivo. Int. J. Radiat. Oncol. Biol. Phys. 20, 525– 530. Smith, J.M., 1958. Prolongation of the life of Drosophila subobscura by a brief exposure of adults to a high temperature. Nature 181, 496– 497. Tatar, M., Khazaeli, A.A., Curtsinger, J.W., 1997. Chaperoning extended life. Nature 390, 30.
Effect of mild whole body hyperthermia on cytotoxic action of N-methyl-N%-nitro-N-nitrosoguanidine in Swiss mice Raghavendra S. Bagewadikar *, Manohar S. Patil, Asifa K. Zaidi, Mylvaganan Subramanian, Gangadhar S. Kaklij Radiation Biology Di6ision, Bhabha Atomic Research Centre, Mumbai 400085, India Received 22 August 2000; received in revised form 22 January 2001; accepted 22 January 2001
Abstract Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a modifier of N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) cytotoxicity in mice. Adult Swiss male mice, 6 – 8 weeks old, weighing about 25 g were exposed to mild WBH (39°C, 1 h) in a precision temperature controlled environmental chamber maintained at 50–60% relative humidity. Twenty-four hours after treatment, animals were administered with different doses of MNNG either by intraperitoneal (i.p.) injections or by feeding through drinking water and were monitored for survival. The studies revealed that the exposure of animals to mild WBH, 24 h prior to MNNG administration results in an increase in survival and recovery in mean body weight compared with those administered with MNNG only. This suggests that prior WBH treatment can effectively reduce the MNNG cytotoxicity in mice. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Whole body hyperthermia; MNNG; Cytotoxicity
1. Introduction In recent years much attention has been focused on protecting living organisms from harmful effects of radiations and from toxic effects of certain environmental and chemotherapeutic agents. Recent studies from our laboratory have shown that the exposure of mice to mild whole body hyperthermia (WBH) at 40°C for 1, 20 or 48 h prior to total body irradiation (TBI) with lethal * Corresponding author. Fax: 91225519613. E-mail address: [email protected] (R.S. Bagewadikar).
dose of g-rays afford significant protection as assessed by survival (Patil et al., 1996). Similar WBH induced radioprotection has been reported by Shen et al. (1991). WBH has also been shown to enhance the antitumour activity of chemotherapeutic agents such as carboplatin, and tumour necrosis factor (TNF) without toxic effects on normal tissues (Sakaguchi et al., 1994). This implies that mild WBH may also protect against toxicity of certain chemicals. N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) is known to mimic the effects of radiation such as induction of mutations (Mandel and
0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 3 1 6 - 2
64
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Greenberg, 1960; Adelberg et al., 1965), cancer (Schoental, 1996), chromosomal aberrations (Gichner et al., 1963) and cell death. It is an alkylating direct acting N-nitroso carcinogen and its mechanism of action is well studied in our laboratory (Bhattacharya and Bagewadikar, 1986; Bagewadikar and Bhattacharya, 1992). It is also highly toxic and shown to have growth inhibitory action on tumour cells (Aujard and Trincal, 1983). In view of this, it was considered of interest to ascertain whether mild WBH pre treatment will modulate MNNG induced cytotoxicity in mice. In the present communication, we report for the first time that mild WBH treatment when given 24 h prior to MNNG administration can effectively reduce MNNG toxicity in mice.
2. Materials and methods
2.1. Animals Syngenic Swiss mice, 6 – 8 weeks old, weighing approximately 25 g, maintained on stock laboratory diet and water ad libitum, were used. The use of animals was approved by the Institutional Animal Ethics Committee (IAEC) and the guidelines for the care of the animals were strictly followed throughout these studies.
2.3. Treatment of animals with MNNG In the first set of experiments MNNG (Fluka A.G. Switzerland) was administered through drinking water (1 mg/ml) to groups of mice (10 mice per group) for 1 and 2 weeks, 24 h after exposure to mild WBH. The control mice without WBH treatment were also fed same concentration of MNNG through drinking water. MNNG solution was supplied fresh every alternate day. Consumption of MNNG in drinking water was measured daily. After 1 and 2 weeks of MNNG treatment, the animals were monitored for their survival. In the second set of experiments, untreated and WBH treated mice received different concentrations of MNNG in saline (80, 120, 160 mg/kg body weight) by repeated i.p. injections 24 h after WBH treatment and were monitored for survival. The weight of each surviving animal was recorded periodically. The dead animals were subjected to postmortem and abdominal and thoracic viscera were examined grossly for deformities and solid tumours in organs.
2.4. Statistical analysis Results are expressed as mean9standard deviation (S.D.). Statistical significance was determined by use of the Log Rank test for survival.
2.2. Whole body hyperthermia treatment 3. Results Just before exposure to WBH, mice were injected intraperitoneally (i.p.) 1 ml of physiological saline (25°C) to compensate the water loss during hyperthermic treatment. For WBH treatment, unanaesthetised mice in groups of 10 were exposed to 39°C and relative humidity of 50 – 60% for 1 h in precision temperature controlled environmental chamber. The rectal temperature before, during and after WBH treatment was measured by a thermocouple probe. The body temperature raised to 40°C within 10 min of exposure and remained unchanged till the period of treatment. Temperature came down to a normal body temperature within 10 min after treatment.
The amount of MNNG consumed per group (10 mice per group) was calculated from daily water intake. Mice in WBH treated group consumed more water compared with untreated group. The amount of MNNG received after 1 week of feeding by untreated and WBH treated group was 86 and 123 mg, respectively. The results depicted in Fig. 1 show the effect of mild WBH on survival of mice against MNNG toxicity. It is evident from the figure that 90% of untreated mice fed with MNNG via drinking water (1 mg/ml for 1 week) died within 36 weeks. However, 50% of the mice exposed to mild WBH, 24 h prior to MNNG feeding survived for more
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
than 52 weeks even after receiving a high dose (43% higher dose) compared with WBH untreated group. Incidentally there was no difference in survival pattern between untreated and WBH treated animals when MNNG was fed for 2 weeks (results not shown). These results indicate that prior WBH treatment results in considerable reduction in MNNG induced toxicity in mice. Postmortem analysis of animals showed enlargement of spleen (Fig. 2a), liver, lymphnodes in most of the cases and solid tumours of lung (Fig. 2b) and small intestine (Fig. 2c) in animals that survived more than 40 weeks from MNNG treated group. This was not evident in WBH pre-treated group indicating that mild WBH protects animals from MNNG induced toxicity when given 24 h prior to MNNG administration. Since the feeding experiments were found to be extremely time consuming and also the dose of MNNG received by each animal was not uniform, the second set of experiments was carried out by i.p. administration of MNNG to mice. Results in Fig. 3 show the dose response of i.p. administered MNNG on survival of mice. It can be seen from the figure that MNNG was highly toxic at the doses of 120 –160 mg/kg body weight. None of the animals survived for more than 10 days after
Fig. 1. Effect of whole body hyperthermia on survival of mice against MNNG toxicity. Mice were given MNNG via drinking water (1 mg/ml, for 1 week) 24 h after WBH and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group. Statistical analysis, MNNG versus MNNG 24 h after WBH P B 0.1.
65
MNNG administration. However, 40% animals survived for more than 120 days at the dose of 80 mg/kg body weight. The next set of experiments on effect of WBH on MNNG toxicity was, therefore, carried out using 80 mg/kg body weight as a test dose. Results in Fig. 4 indicate that when MNNG was administered immediately before or after WBH, the survival of animals was decreased significantly. However, the animals which were given WBH 24 h prior to MNNG administration showed significant increase in survival compared with those who received MNNG only. This is corroborated by the recovery in mean body weight in WBH treated animals compared with those administered with MNNG only (Fig. 5). The data strongly suggest that prior WBH treatment results in protection against MNNG toxicity in mice.
4. Discussion The most interesting finding on WBH induced radioprotection (Shen et al., 1991; Patil et al., 1996) in mice prompted us to investigate whether prior WBH will also afford protection against toxic effects of radiomimetic compounds like MNNG. Our results reported in this communication serve to show that mild WBH (39°C, 1 h) treatment given 24 h prior to MNNG administration can afford significant protection in terms of survival of mice against MNNG cytotoxicity. The protective effect depends upon the time of MNNG administration after WBH treatment. The WBH induced increase in survival after MNNG administration corresponds very well with the recovery in mean body weight of WBH treated animals compared with those who received MNNG only. Furthermore, WBH induced reduction in MNNG cytotoxicity is confirmed by two independent modes of MNNG administration. The molecular mechanisms underlying the WBH induced reduction in MNNG toxicity are far from clear. Several recent studies have shown that heat shock proteins (HSPs) can protect cells from wide variety of stresses such as oxidant
66
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Fig. 2. Postmortem analysis of mice treated with MNNG: showing (a) enlarged spleen (b) solid tumor of lung (c) solid tumor of small intestine, from mice killed at 40 weeks after MNNG feeding via drinking water (1 mg/ml for 1 week). Small spleen in (a) is from mice treated with WBH + MNNG. No tumours were found in WBH+MNNG treated mice.
radicals or endotoxins (Hartl, 1996; Kiang and Tsokos, 1998). Frossard (1999) has reported excellent correlation between increased level of WBH induced HSP 70 expression in rat pancreas and increase in time to death in rats exposed to hyperthermia, when control and hyperthermia treated animals were killed using rapid asphyxia method with 100% CO2. Induction of HSPs in animals by WBH protects the brain and heart from tissue injury induced by ischemia (Liu et al., 1992; Brown and Sharp, 1999; Carroll and Yellon, 1999; Ohtsuka and Hata, 2000). Similarly, increased expression of HSPs results in extended life span in nematoda and the fruit fly (Smith, 1958; Lithgow et al., 1995; Tatar et al., 1997). It has also been reported that administration of cytokines such as transforming growth factor-b (TGF-b) exhibit myloprotection during chemotherapy with 5-flurouracil and cyclophosphamide (Lemoli et al., 1992; Carlino and Gregory, 1993; Grzegorzewski et al., 1994; Kekow et al., 1995). WBH treatment is known to increase
levels of various cytokines including TGF-b (Robins et al., 1995; Kekow et al., 1995). Kekow et al. (1995) have reported that transient increase
Fig. 3. Dose response of intraperitoneally administered MNNG on survival of mice. Mice were exposed to WBH (39°C for 1 h) 24 h prior to MNNG administration at 80 – 160 mg/kg bodyweight and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group.
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Fig. 4. Survival of mice treated with MNNG after WBH treatment. Mice were intraperitoneally administered with MNNG (80 mg/kg body weight), immediately before, immediately after and 24 h after WBH (39°C for 1 h) and were monitored for survival. The data are representative of three independent experiments involving 10 mice in each group. Statistical analysis, MNNG versus MNNG immediately before WBH P B 0.1; MNNG versus MNNG immediately after WBH PB 0.001; MNNG versus MNNG 24 h after WBH P B 0.05.
in TGF-b1 and TGF-b2 under WBH might play a role in protecting bone marrow from toxic effects of myelosuppressive chemotherapy. Thus, from the voluminous information available on WBH effects in biological systems, it would seem that induction of HSPs and certain cytokines after WBH might play an important role in protection against toxic effects of MNNG. This aspect is being currently investigated in our laboratory. In conclusion, our results seem to suggest that appropriate scheduling of WBH treatment and MNNG administration results in significant reduction in toxicity of even a highly potent genotoxic and proteotoxic chemical like MNNG. This implies that WBH may have a great potential in protecting against toxic effects of drug treatments and also in reducing the toxicity of chemotherapeutic agents used in cancer chemotherapy.
Acknowledgements We are thankful to V.G. Khankal and K.S. Gosavi for their excellent technical assistance.
67
Fig. 5. Effect of WBH on mean body weight of mice administered with MNNG (80 mg/kg body weight). The data represent the mean of three independent experiments.
References Adelberg, E.A., Mandel, M., Chen, G.C.C., 1965. Optimal conditions for mutagenesis by MNNG in E. Coli K12. Biochem. Biophys. Res. Commun. 18, 788– 795. Aujard, C., Trincal, G., 1983. The effects of N-methyl-N%-nitro-N-nitrosoguanidine on KB cells. 1.Growth inhibition and cell killing in relation to inhibition of macromolecular synthesis. Chem. Biol. Interact. 44, 79 – 93. Bagewadikar, R.S., Bhattacharya, R.K., 1992. Effect of Nmethyl-N%-nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach. J. Biochem. Toxicol. 7, 19 – 24. Bhattacharya, R.K., Bagewadikar, R.S., 1986. Functional modification of rat liver ribosomes by the in vitro action of N-methyl-N%-nitro-N-nitrosoguanidine. Chem. Biol. Interact. 57, 235– 251. Brown, I.R., Sharp, F.R., 1999. The cellular stress response in brain. In: Latchman, D.S. (Ed.), Stress Proteins. Springer, Berlin, pp. 243– 263. Carlino, J.A., Gregory, R.W., 1993. TGF-b2 decreases chemotherapy induced mortality in mice. J. Cell Biochem. 17, 61. Carroll, R., Yellon, D.M., 1999. Heat stress proteins and their relationship to myocardial protection. In: Latchman, D.S. (Ed.), Stress Proteins. Springer, Berlin, pp. 265– 279.
68
R.S. Bagewadikar et al. / Toxicology Letters 121 (2001) 63–68
Frossard, J.L., 1999. Heat shock protein 70 (HSP 70) prolongs survival in rats exposed to hyperthermia. Eur. J. Clin. Invest. 29, 561– 562. Gichner, T., Michaelis, A., Rieger, R., 1963. Radiomimetic effects of 1-methyl-3-nitro-1-nitrosoguanidine in Vicia faba. Biochem. Biophys. Res. Commun. 11, 120–124. Grzegorzewski, K., Ruscetti, F.W., Usui, N., et al., 1994. Recombinant transforming growth factor b1 and b2 protect mice from acutely lethal doses of 5-fluorouracil and doxorubicin. J. Exp. Med. 180, 1047–1057. Hartl, F.U., 1996. Molecular chaperones in cellular protein folding. Nature 381, 571–579. Kekow, J., Wiedemann, G.J., Katschinski, D.M., Kisro, J., Geisler, J., Wagner, T., Szymkowiak, C., 1995. Whole body hyperthermia increases plasma levels of transforming growth factor b. Int. J. Oncol. 7, 1427–1432. Kiang, J.G., Tsokos, G.C., 1998. Heat shock protein 70 kDa: molecular biology, biochemistry, physiology. Pharmacol. Ther. 80, 183– 201. Lemoli, R.M., Strife, A., Clarkson, B.D., et al., 1992. TGF-b3 protects normal human hemopoietic progenitor cells treated with 4-hydroperoxy cyclophosphamide in vitro. Exp. Hematol. 20, 12 – 52. Lithgow, G.S., White, T.M., Melov, S., Johnson, T.E., 1995. Thermotolerance and extended life span conferred by single gene mutations and induced by thermal stress. Proc. Natl. Acad. Sci. USA 92, 7540–7544. Liu, X., Engelman, R.M., Moraru, I.I., Rousou, J.A., Flack, J.E., Deaton, D.W., Moulik, N., Das, D.K., 1992. Heat shock: a new approach for myocardial preservation in cardiac surgery. Circulation 86, 11358–11363.
.
Mandel, J.D., Greenberg, J., 1960. A new chemical mutagen for bacteria, 1-methyl-3-nitro-1-nitrosoguanidine. Biochem. Biophys. Res. Commun. 3, 575– 577. Ohtsuka, K., Hata, M., 2000. Molecular chaperone function of mammalian HSP 70 and HSP 40: a review. Int. J. Hyperthermia 16, 231– 245. Patil, M.S., Kaklij, G.S., Poduval, T.B., Singh, B.B., 1996. Radioprotective effect of whole body hyperthermia on mice exposed to lethal doses of total body gamma irradiation. Ind. J. Exp. Biol. 34, 842– 844. Robins, H.I., Kutz, M., Wiedemann, G.J., Katschinski, D.M., Paul, D., Grosen, E., Tiggelaar, C.L., Spriggs, D., Gillis, W., d’Oleire, F., 1995. Cytokine induction by 41.8°C whole body hyperthermia. Cancer lett. 97, 195– 201. Sakaguchi, Y., Makino, M., Kaneko, T., Stephens, L.C., Strebel, F.R., Danhauser, L.L., Jenkins, G.N., Bull, J.M.C., 1994. Therapeutic efficacy of long duration-low temperature whole body hyperthermia when combined with tumor necrosis factor and carboplatin in rats. Cancer Res. 54, 2223– 2227. Schoental, R., 1996. Carcinogenic activity of N-methyl-N%-nitro-N-nitrosoguaninine. Nature 209, 726– 727. Shen, R.N., Hornback, N.B., Shidnia, H., Wu, B., Lu, L., Broxeyer, H.E., 1991. Whole body hyperthermia: a potent radioprotector in vivo. Int. J. Radiat. Oncol. Biol. Phys. 20, 525– 530. Smith, J.M., 1958. Prolongation of the life of Drosophila subobscura by a brief exposure of adults to a high temperature. Nature 181, 496– 497. Tatar, M., Khazaeli, A.A., Curtsinger, J.W., 1997. Chaperoning extended life. Nature 390, 30.