- Email: [email protected]
− mice exposed to diethylstilboestrol in the diet
70
Abstracts
The apoptotic response was both time- and dose-dependent. Intact glucosinolates, including sinigrin, gluconapin and progoitrin, have previously been shown not to be cytotoxic to HT-29 cells when administered individually (Nastruzzi et al., 1996). This indicates that the combination of glucosinolates in the extract may have a synergistic cytotoxic effect. Our preliminary investigations have also indicated that the HT-29 cells may metabolise the glucosinolates to their more active hydrolysis products (e.g. isothiocyanates), which are highly cytotoxic to HT-29 cells. Further investigations will determine the importance of glucosinolate hydrolysis by the cells. These data show for the first time that treatment with intact glucosinolates extracted from Brussels sprouts inhibits the growth of colon cancer cells in vitro by activating apoptosis. This represents a potential mechanism for the chemoprotective activity of cruciferous vegetables. Chung, F.-L., Kelloff, G., Steele, V., Pittman, B., Zang, E., Jiao, D., Rigotty, J., Choi, C.-I., Rivenson, I., 1996. Cancer Res. 56, 772-778. Musk, S.R.R., Stephenson, P., Smith, T.K., Stening, P., Fyfe, D., Johnson, I.T., 1995. Nutr. Cancer 24, 289-298. Nastruzzi, I., Cortesi, R., Esposito, E., Menegiatti, E., Leoni, O., Ion, R., Palmieri, S., 1996. J. Agric. Food Chem. 44, 1014-21. Yuan, J.-M., Gago-Dominguez, M., Castelao, E., Hankin, J.H., Ross, R.K., Yu, M.C., 1998. Int. J. Cancer, 77, 211-216.
The Incidence of Primary Tumours in XPA -I - and XPA - ’ - p53 + Iin the Diet
Mice Exposed to Diethylstilboestrol
Peter A. McAnulty and Mikala Skydsgaard SCANTOX, Hestehavevej 36A, Ejby, DK-4623 Lille Skensved, Denmark Several transgenic strains of mice are under investigation as possible alternative models for carcinogenicity testing as part of a programme organised by ILSI-HESI. Among these are XPA-‘mice (XPA) and ~53 +I- mice (~53). XPA mice have a homozygous deletion of the XPA gene, and the protein coded by this gene is essential for the detection and repair of DNA damage. ~53 mice have one allele knocked out of the ~53 gene, often referred to as a tumour-suppressor gene. We have evaluated the effect of diethylstilboestrol (DES) in both XPA mice, and double transgenic mice with both a homozygous XPA knock-out and a hemizygous ~53 knock-out (XPA/p53). DES is considered to be a non-mutagenic carcinogen, exerting its carcinogenic effect in humans by hormonal influences. XPA mice (15 males and 15 females per group) were fed diets containing DES for nine months at dietary concentrations of 0, 100, 300 and 1500 ppb. XPA/p53 mice and wild-type mice (WT) of the same group sizes were fed DES at 0 or 1500 ppb. At termination of the study, a full histopathological examination was performed on all animals. However, this report is restricted to the incidence of primary tumours in the three types of mice at the 0 and 1500 ppb dietary concentrations: The incidence of pituitary adenomas appeared to be slightly increased in the XPA mice compared with the double transgenic and wild type mice, although this was not statistically significant, and almost certainly unrelated to the genetic status of the animals. The most notable finding was the incidence of osteosarcomas in the XPA/p53 mice. Osteosarcomas occur as background in ~53 mice, but their incidence was not increased in a 6-month study with DES (R.T. Bunch and C. Alden, personal communication), compared with the 9 months used in this study. This suggests that a 6-month study in ~53 mice may not be sufficient for detection of carcinogens of this type.
71
Abstracts
DES (ppb)
0
Primary tumours Testicular interstitial cell adenoma Osteosarcoma Lymphoma Pituitary adenoma Mammary carcinoma Phaeochromocytoma Brain glioma Cervical fibrosarcoma Total of tumourbearing animals
Transcriptional
XPA/p53 0
XPA 1500
M 0
F
0 0 0 0 0 0
M 0
F
0 0 3 0
0 0 0
0
0
0
0
0
0 0 3
0
M 1
F
0 0 0 0
1 1 5
0
0 0 0
Response to Glutathione
8
0
0 0 1 0 0 0 1
WT 0
1500 M 4
F
4 2 0 2 220022 0 1 0 1 0 1 12 7
1500
M 0
F
0 0
0 0
0 0 0
M
F
1
0 0 0 0 0
0 1
0 0 4
0 0 0 0 0 0 2
Depletion
H. Powell, N.R. Kitteringham, Y. Clement, C. O’Donnell, C. Goldring, M. Pirmohamed. B.K. Park Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, UK Chemical and oxidative stress, characterised by depletion of reduced glutathione (GSH), results in a cellular response aimed at restoring GSH levels. Part of this response involves enhanced expression of the dimeric enzyme y-glutamylcysteine synthetase (y-GCS), the rate-limiting enzyme of GSH synthesis. Transcription of y-GCS subunit genes is in part regulated by the transcription factor AP-1 (Mulcahy and Gipp, 1995), a protein formed from dimers of the Fos and Jun families of proteins (Angel and Karin, 1991). with Jun homodimers being more active than Fos/Jun heterodimers (Sekhar et al., 1997). The stimulus for up-regulation of antioxidant genes in response to GSH depletion remains unresolved, with GSH levels themselves or the accumulation of GSH-conjugates being postulated (Shelton et al., 1986). We have therefore compared y-GCS mRNA and protein levels and factors regulating its expression following administration of either diethyl maleate (DEM), which depletes GSH by conjugate formation, or buthionine sulphoximine (BSO), which depletes GSH through inhibition of y-GCS, without conjugate formation. In addition, we have studied the expression of the alpha chain of the nascent polypeptide-associated complex (c(-NAC), a recently discovered transcriptional co-activator of AP-1 in differentiating bone that preferentially enhances the activity of Jun homodimers (St-Arnaud and Quelo, 1998). Male CD-l mice (30-40 g; n = 6 per group) were administered a single i.p. dose of either DEM (4.7 mmol/kg) or BSO (7.2 mmol/kg in saline). At 0.5, 1, 2, 3, 5 and 24 h, hepatic GSH was determined. Hepatic y-GCS, c-fos, c-jun and alpha-NAC mRNAs were measured by RT-PCR; AP-1 DNA-binding activity was determined by the electrophoretic mobility shift assay: and y-GCS and alpha-NAC protein levels were determined by Western blotting. Hepatic GSH depletion of 60-70% was observed within 2 h following the administration of both DEM and BSO, with levels returning to pre-treatment values by 24 h. DEM treatment led to an increase in both y-GCS mRNA (3-fold) and protein levels (1.8-fold), which reflected concomitant rises in c-fos ancl c-jun mRNA levels, and AP-1 DNA binding activity. In contrast, no significant changes in either the transcription or expression of y-GCS were observed following the administration of BSO, despite
Abstracts
The apoptotic response was both time- and dose-dependent. Intact glucosinolates, including sinigrin, gluconapin and progoitrin, have previously been shown not to be cytotoxic to HT-29 cells when administered individually (Nastruzzi et al., 1996). This indicates that the combination of glucosinolates in the extract may have a synergistic cytotoxic effect. Our preliminary investigations have also indicated that the HT-29 cells may metabolise the glucosinolates to their more active hydrolysis products (e.g. isothiocyanates), which are highly cytotoxic to HT-29 cells. Further investigations will determine the importance of glucosinolate hydrolysis by the cells. These data show for the first time that treatment with intact glucosinolates extracted from Brussels sprouts inhibits the growth of colon cancer cells in vitro by activating apoptosis. This represents a potential mechanism for the chemoprotective activity of cruciferous vegetables. Chung, F.-L., Kelloff, G., Steele, V., Pittman, B., Zang, E., Jiao, D., Rigotty, J., Choi, C.-I., Rivenson, I., 1996. Cancer Res. 56, 772-778. Musk, S.R.R., Stephenson, P., Smith, T.K., Stening, P., Fyfe, D., Johnson, I.T., 1995. Nutr. Cancer 24, 289-298. Nastruzzi, I., Cortesi, R., Esposito, E., Menegiatti, E., Leoni, O., Ion, R., Palmieri, S., 1996. J. Agric. Food Chem. 44, 1014-21. Yuan, J.-M., Gago-Dominguez, M., Castelao, E., Hankin, J.H., Ross, R.K., Yu, M.C., 1998. Int. J. Cancer, 77, 211-216.
The Incidence of Primary Tumours in XPA -I - and XPA - ’ - p53 + Iin the Diet
Mice Exposed to Diethylstilboestrol
Peter A. McAnulty and Mikala Skydsgaard SCANTOX, Hestehavevej 36A, Ejby, DK-4623 Lille Skensved, Denmark Several transgenic strains of mice are under investigation as possible alternative models for carcinogenicity testing as part of a programme organised by ILSI-HESI. Among these are XPA-‘mice (XPA) and ~53 +I- mice (~53). XPA mice have a homozygous deletion of the XPA gene, and the protein coded by this gene is essential for the detection and repair of DNA damage. ~53 mice have one allele knocked out of the ~53 gene, often referred to as a tumour-suppressor gene. We have evaluated the effect of diethylstilboestrol (DES) in both XPA mice, and double transgenic mice with both a homozygous XPA knock-out and a hemizygous ~53 knock-out (XPA/p53). DES is considered to be a non-mutagenic carcinogen, exerting its carcinogenic effect in humans by hormonal influences. XPA mice (15 males and 15 females per group) were fed diets containing DES for nine months at dietary concentrations of 0, 100, 300 and 1500 ppb. XPA/p53 mice and wild-type mice (WT) of the same group sizes were fed DES at 0 or 1500 ppb. At termination of the study, a full histopathological examination was performed on all animals. However, this report is restricted to the incidence of primary tumours in the three types of mice at the 0 and 1500 ppb dietary concentrations: The incidence of pituitary adenomas appeared to be slightly increased in the XPA mice compared with the double transgenic and wild type mice, although this was not statistically significant, and almost certainly unrelated to the genetic status of the animals. The most notable finding was the incidence of osteosarcomas in the XPA/p53 mice. Osteosarcomas occur as background in ~53 mice, but their incidence was not increased in a 6-month study with DES (R.T. Bunch and C. Alden, personal communication), compared with the 9 months used in this study. This suggests that a 6-month study in ~53 mice may not be sufficient for detection of carcinogens of this type.
71
Abstracts
DES (ppb)
0
Primary tumours Testicular interstitial cell adenoma Osteosarcoma Lymphoma Pituitary adenoma Mammary carcinoma Phaeochromocytoma Brain glioma Cervical fibrosarcoma Total of tumourbearing animals
Transcriptional
XPA/p53 0
XPA 1500
M 0
F
0 0 0 0 0 0
M 0
F
0 0 3 0
0 0 0
0
0
0
0
0
0 0 3
0
M 1
F
0 0 0 0
1 1 5
0
0 0 0
Response to Glutathione
8
0
0 0 1 0 0 0 1
WT 0
1500 M 4
F
4 2 0 2 220022 0 1 0 1 0 1 12 7
1500
M 0
F
0 0
0 0
0 0 0
M
F
1
0 0 0 0 0
0 1
0 0 4
0 0 0 0 0 0 2
Depletion
H. Powell, N.R. Kitteringham, Y. Clement, C. O’Donnell, C. Goldring, M. Pirmohamed. B.K. Park Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, UK Chemical and oxidative stress, characterised by depletion of reduced glutathione (GSH), results in a cellular response aimed at restoring GSH levels. Part of this response involves enhanced expression of the dimeric enzyme y-glutamylcysteine synthetase (y-GCS), the rate-limiting enzyme of GSH synthesis. Transcription of y-GCS subunit genes is in part regulated by the transcription factor AP-1 (Mulcahy and Gipp, 1995), a protein formed from dimers of the Fos and Jun families of proteins (Angel and Karin, 1991). with Jun homodimers being more active than Fos/Jun heterodimers (Sekhar et al., 1997). The stimulus for up-regulation of antioxidant genes in response to GSH depletion remains unresolved, with GSH levels themselves or the accumulation of GSH-conjugates being postulated (Shelton et al., 1986). We have therefore compared y-GCS mRNA and protein levels and factors regulating its expression following administration of either diethyl maleate (DEM), which depletes GSH by conjugate formation, or buthionine sulphoximine (BSO), which depletes GSH through inhibition of y-GCS, without conjugate formation. In addition, we have studied the expression of the alpha chain of the nascent polypeptide-associated complex (c(-NAC), a recently discovered transcriptional co-activator of AP-1 in differentiating bone that preferentially enhances the activity of Jun homodimers (St-Arnaud and Quelo, 1998). Male CD-l mice (30-40 g; n = 6 per group) were administered a single i.p. dose of either DEM (4.7 mmol/kg) or BSO (7.2 mmol/kg in saline). At 0.5, 1, 2, 3, 5 and 24 h, hepatic GSH was determined. Hepatic y-GCS, c-fos, c-jun and alpha-NAC mRNAs were measured by RT-PCR; AP-1 DNA-binding activity was determined by the electrophoretic mobility shift assay: and y-GCS and alpha-NAC protein levels were determined by Western blotting. Hepatic GSH depletion of 60-70% was observed within 2 h following the administration of both DEM and BSO, with levels returning to pre-treatment values by 24 h. DEM treatment led to an increase in both y-GCS mRNA (3-fold) and protein levels (1.8-fold), which reflected concomitant rises in c-fos ancl c-jun mRNA levels, and AP-1 DNA binding activity. In contrast, no significant changes in either the transcription or expression of y-GCS were observed following the administration of BSO, despite