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Mutagenicity of N-nitroso-N-ethylurea (ENU) in the transgenic Muta®Mouse
184 made from control samples (DMSO) or from samples obtained following treatment of cells with BHA '(1 /~g/ml medium) or TPA (0.1 # g / m l medium) for 6 days. We have demonstrated in all samples, following standardised silver staining, a number of bands representing proteins in the molecular weight range 35-67 kD, corresponding closely to the range for keratins. A low-molecular-weight band (approx. 36 kD) was strongly expressed in control samples but was almost absent from samples treated with BHA or TPA. Also, 2 high-molecular-weight bands (approx. 58.5 kD and 66-67 kD) in the treated samples were almost invisible in lanes carrying control samples. The protein changes were identical in cells treated with BHA or TPA, despite the fact that our previous assays for differentiation have shown a much greater positive influence exerted by TPA. We suggest that these changes may indicate increased production of high-molecular-weight, differentiation-specific keratins on exposure to these compounds, via an epigenetic mechanism, not involving protein kinase C. The equivalent action of these 2 compounds may further suggest that these 2 classes of compound may both be described by the common term non-genotoxic carcinogen.
37 Burlinson, B., S. Morriss, D. Gatehouse and D.J. Tweats, Genetic and Reproductive Toxicology Department, Glaxo Group Research, Ware (Great Britain)
Comparative studies on the gastric acid inhibiting drugs (ioxtidine, ranitidine, cimetidine and omeprazole) using the in vivo stomach U D S assay Loxtidine and omeprazole are acid-suppressants proposed for the treatnient of gastric ulcers. The former is a potent unsurmountable histamine H2-antagonist, whilst the latter is a proton-pump inhibitor specifically inhibiting the H + / K + ATPase enzyme system at the surface of the parietal cell. Both drugs have been associated with
the appearance of gastric carcinoid tumours following 10ng-term oral administration to rats. Neither drug has exhibited genotoxicity in vitro or in vivo using a range of short-term tests. Often false negative results can occur if the organotropic nature of the drug is not considered. For this reason the genotoxic potential of both drugs was determined in the possible target tissue (gastric mucosa) using an in vivo stomach Unscheduled DNA synthesis (UDS) assay. For comparative purposes the HE-antagonists, ranitidine and cimetidine were also evaluated. The drugs were administered orally to rats at doses up to 100-fold higher than the likely therapeutic dose. After 14 h the stomachs were removed, the DNA of the mucosal cells extracted (not stem cells), and the uptake of tritiated thymidine (injected subcutaneously 1 h previously) was assessed scintillo-metrically. None of the HE-antagonists (loxtidine, ranitidine and cimetidine) induced UDS in the rat gastric mucosa. However, omeprazole induced significant and reproducible increases in UDS. These results were not due to any trophic effects (i.e. stimulation of cell division) since the administration of pentagastrin was negative in this assay. It is concluded that omeprazole has the potential to act as an initiating carcinogen whereas the other antisecretory agents examined do not. The relevance of these findings to the occurrence of carcinoid tumours within the rat stomach is not known.
38 Hoorn, A.J.W. i, L.L. Custer and J.A. Gossen 2, 1 Hazleton Laboratories America, Inc., Kensington, MD (U.S.A.) and 2TNO Institute for Experimental Gerontology, Rijswijk (The Netherlands) Mutagenicity of N-nitroso-N-ethylurea (ENU) in the transgenic Muta®Mouse The transgenic Muta*Mouse, developed at TNO, carrying 80 copies of the bacterial LacZ gene per cell as target for mutagenesis, is presently being evaluated as a practical system for assaying
185 gene mutations in vivo. The integrated vectors were rescued from genomic DNA of different tissues with an in vitro lambda packaging preparation and the phages were propagated in an E. coli C LacZ- strain. In this first study of several model mutagens, ENU was injected i.p. to 8-10week-old male mice. Both acute (100 and 250 mg/kg) and subchronic (5 daily injections of 10 and 50 mg/kg) dosing regimens were used, and the animals were killed 3, 7 and 10 days after the last injection of the test chemical. The liver, spleen, kidneys, testes, brain and bone marrow were dissected, frozen in liquid nitrogen, and kept at - 8 0 °C until analyzed. For each group, 3 male mice were used while also 3 males, serving as solvent controls, were killed 2 days after an injection with dimethyl sulfoxide. Background mutant frequencies were approximately 0.6 × 10 -5 and 1.7 × 10 -5 for the testis and bone marrow, respectively. ENU induced dose-related increases in mutant frequency ranging from 27 times the background for the testis up to approximately 50 times the spontaneous value for the bone marrow. These maximum increases were observed 7 days after the last dose for both acute and subchronic treatments of the testis and bone marrow.
high levels of ATase expression (greater than 30 f m / m g protein versus < 2 f m / m g protein in parent vector-transfected control cells). Microinjection of a 4.2-kb fragment of this vector into B6D2F2 embryos and implantation of survivors into pseudopregnant females has so far generated 35 offspring. Southern analysis of tailtip DNA has shown that 11 of the offspring are transgenic for the human ATase, between 1 and at least 50 copies of the gene being detected. Restriction endonuclease analysis using isoschizomers MspI and HpalI indicate that in some transgenes corresponding (CCGG) sites in the human gene are undermethylated, suggesting that the gene may express in these animals.
39
Effects of SO2 and NOx on the metabolic activation of N-nitrosodimethylamine and benzolalpyrene
Potter, P.M., C.Y. Fan, J.A. Rafferty, A.J. Watson, P.S. Searle 1, p.j. O'Connor and G.P. Margison, Cancer Research Campaign Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Manchester M20 9BX and 1 Cancer Research Campaign Department of Cancer Studies, University of Birmingham, Birmingham (U.K.)
Cytosine methylation patterns in human O 6 - 8 1 kyiguanine-DNA-alkyltransferase transgenic mice A truncated human O6-alkylguanine-DNA-aikyltransferase (ATase) cDNA was ligated into an expression vector under the control of the mouse metallothionein-1 gene promoter and upstream of the human growth hormone gene. Transfection of this construct into human cells resulted in very
40 Pasquini, R. 1, S. Monarca 2, R. Klein 3, p. Schrnezer 3, W.J. Zeller 3, R. Hermann 3, B.L. Pool-Zobel 3 and U. Moretti 1 1 Dept. of Hygiene, University of Perugia, 2 Chair of Environmental Health, 0niversity of Brescia (Italy) and 3 Institute for Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg (F.R.G.)
In the course of a study aimed at elucidating combination effects of air pollutants and carcinogens, we have demonstrated in mammalian cells that SO2 may decrease the genotoxic activity of N-nitrosodimethylamine (NDMA) and NO~ that of benzo[a]pyrene (BP). Proposed mechanisms were energy depletion by SO 2 (Pool et al. (1988a) Carcinogenesis, 9), influence on DNA repair by NO x (Zeller et al. (1988) KFK-PEF, 35), and effects on carcinogen-metabolizing enzymes. Indeed we found that inhaled NO x decreased arylhydrocarbon hydroxylases (AHH), NDMA demethylases (NDMA-D) and glutathione transferases (GST) in the liver and GST in the lung, and SO2 increased NDMA-D in the liver and decreased GST in the lung (Pool et al. (1988b)
37 Burlinson, B., S. Morriss, D. Gatehouse and D.J. Tweats, Genetic and Reproductive Toxicology Department, Glaxo Group Research, Ware (Great Britain)
Comparative studies on the gastric acid inhibiting drugs (ioxtidine, ranitidine, cimetidine and omeprazole) using the in vivo stomach U D S assay Loxtidine and omeprazole are acid-suppressants proposed for the treatnient of gastric ulcers. The former is a potent unsurmountable histamine H2-antagonist, whilst the latter is a proton-pump inhibitor specifically inhibiting the H + / K + ATPase enzyme system at the surface of the parietal cell. Both drugs have been associated with
the appearance of gastric carcinoid tumours following 10ng-term oral administration to rats. Neither drug has exhibited genotoxicity in vitro or in vivo using a range of short-term tests. Often false negative results can occur if the organotropic nature of the drug is not considered. For this reason the genotoxic potential of both drugs was determined in the possible target tissue (gastric mucosa) using an in vivo stomach Unscheduled DNA synthesis (UDS) assay. For comparative purposes the HE-antagonists, ranitidine and cimetidine were also evaluated. The drugs were administered orally to rats at doses up to 100-fold higher than the likely therapeutic dose. After 14 h the stomachs were removed, the DNA of the mucosal cells extracted (not stem cells), and the uptake of tritiated thymidine (injected subcutaneously 1 h previously) was assessed scintillo-metrically. None of the HE-antagonists (loxtidine, ranitidine and cimetidine) induced UDS in the rat gastric mucosa. However, omeprazole induced significant and reproducible increases in UDS. These results were not due to any trophic effects (i.e. stimulation of cell division) since the administration of pentagastrin was negative in this assay. It is concluded that omeprazole has the potential to act as an initiating carcinogen whereas the other antisecretory agents examined do not. The relevance of these findings to the occurrence of carcinoid tumours within the rat stomach is not known.
38 Hoorn, A.J.W. i, L.L. Custer and J.A. Gossen 2, 1 Hazleton Laboratories America, Inc., Kensington, MD (U.S.A.) and 2TNO Institute for Experimental Gerontology, Rijswijk (The Netherlands) Mutagenicity of N-nitroso-N-ethylurea (ENU) in the transgenic Muta®Mouse The transgenic Muta*Mouse, developed at TNO, carrying 80 copies of the bacterial LacZ gene per cell as target for mutagenesis, is presently being evaluated as a practical system for assaying
185 gene mutations in vivo. The integrated vectors were rescued from genomic DNA of different tissues with an in vitro lambda packaging preparation and the phages were propagated in an E. coli C LacZ- strain. In this first study of several model mutagens, ENU was injected i.p. to 8-10week-old male mice. Both acute (100 and 250 mg/kg) and subchronic (5 daily injections of 10 and 50 mg/kg) dosing regimens were used, and the animals were killed 3, 7 and 10 days after the last injection of the test chemical. The liver, spleen, kidneys, testes, brain and bone marrow were dissected, frozen in liquid nitrogen, and kept at - 8 0 °C until analyzed. For each group, 3 male mice were used while also 3 males, serving as solvent controls, were killed 2 days after an injection with dimethyl sulfoxide. Background mutant frequencies were approximately 0.6 × 10 -5 and 1.7 × 10 -5 for the testis and bone marrow, respectively. ENU induced dose-related increases in mutant frequency ranging from 27 times the background for the testis up to approximately 50 times the spontaneous value for the bone marrow. These maximum increases were observed 7 days after the last dose for both acute and subchronic treatments of the testis and bone marrow.
high levels of ATase expression (greater than 30 f m / m g protein versus < 2 f m / m g protein in parent vector-transfected control cells). Microinjection of a 4.2-kb fragment of this vector into B6D2F2 embryos and implantation of survivors into pseudopregnant females has so far generated 35 offspring. Southern analysis of tailtip DNA has shown that 11 of the offspring are transgenic for the human ATase, between 1 and at least 50 copies of the gene being detected. Restriction endonuclease analysis using isoschizomers MspI and HpalI indicate that in some transgenes corresponding (CCGG) sites in the human gene are undermethylated, suggesting that the gene may express in these animals.
39
Effects of SO2 and NOx on the metabolic activation of N-nitrosodimethylamine and benzolalpyrene
Potter, P.M., C.Y. Fan, J.A. Rafferty, A.J. Watson, P.S. Searle 1, p.j. O'Connor and G.P. Margison, Cancer Research Campaign Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Manchester M20 9BX and 1 Cancer Research Campaign Department of Cancer Studies, University of Birmingham, Birmingham (U.K.)
Cytosine methylation patterns in human O 6 - 8 1 kyiguanine-DNA-alkyltransferase transgenic mice A truncated human O6-alkylguanine-DNA-aikyltransferase (ATase) cDNA was ligated into an expression vector under the control of the mouse metallothionein-1 gene promoter and upstream of the human growth hormone gene. Transfection of this construct into human cells resulted in very
40 Pasquini, R. 1, S. Monarca 2, R. Klein 3, p. Schrnezer 3, W.J. Zeller 3, R. Hermann 3, B.L. Pool-Zobel 3 and U. Moretti 1 1 Dept. of Hygiene, University of Perugia, 2 Chair of Environmental Health, 0niversity of Brescia (Italy) and 3 Institute for Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg (F.R.G.)
In the course of a study aimed at elucidating combination effects of air pollutants and carcinogens, we have demonstrated in mammalian cells that SO2 may decrease the genotoxic activity of N-nitrosodimethylamine (NDMA) and NO~ that of benzo[a]pyrene (BP). Proposed mechanisms were energy depletion by SO 2 (Pool et al. (1988a) Carcinogenesis, 9), influence on DNA repair by NO x (Zeller et al. (1988) KFK-PEF, 35), and effects on carcinogen-metabolizing enzymes. Indeed we found that inhaled NO x decreased arylhydrocarbon hydroxylases (AHH), NDMA demethylases (NDMA-D) and glutathione transferases (GST) in the liver and GST in the lung, and SO2 increased NDMA-D in the liver and decreased GST in the lung (Pool et al. (1988b)