- Email: [email protected]
−-mice by different pathways
S40
Abstracts / Toxicology Letters 180S (2008) S32–S246
as an agonist of the constitutive androstane receptor (CAR) and may also have some agonistic activity at the Ah receptor. doi:10.1016/j.toxlet.2008.06.671 M27 Investigation of the brain distribution of the neuronal cytochrome oxidase activity in adult mice submitted to a subacute benzo(a)pyrene administration Julie Peiffer 1 , Catherine Strazielle 2 , Henri Schroeder 1,∗ 1
URAFPA, INRA UC340, Vandoeuvre les Nancy, France, U724, Vandoeuvre les Nancy, France
2
INSERM
A 10-day subacute administration of benzo(a)pyrene (BaP) has been shown to induce specific behavioural alterations including spatial memory and anxiety, in relation with significant variations of the NR1 and NR2a NMDA receptor subunit expression located in the hippocampus, the temporal cortex, and the frontal cortex. In order to precise the sensitivity of cerebral areas to BaP, the regional distribution of cytochrome oxidase (CO) activity, a mitochondrial enzyme marker for neuronal activation, was investigated in mice treated with BaP. Groups of six mice each were daily injected i.p. with BaP (0.02, 0.2, 2, 20, 200 mg/kg/day) for 10 days. At the end of the period of treatment, the mice were sacrificed, and brains were removed and deep frozen. Thus, frontal sections of 20-m thick were collected from each brain. The CO activity was revealed by histochemistry using the 3–3 diaminobenzidine as substrate, and measured in 68 structures using an image analysis densitometer. The results showed that BaP induced significant increases in CO activity in 43 of the 68 brain areas studied. Over the range of doses injected, the CO activity was significantly increased in mainly limbic structures such as the amygdala, the hippocampus, the lateral septum, some specific hypothalamus and thalamus nuclei, and various cortical areas including the entorhinal, perirhinal, piriform, and cingular cortices. No significant variation was observed in both motor and sensory areas. These findings suggest that BaP induced specific alterations of the cerebral activity in regions that highly correlated with the behavioural disorders previously observed. doi:10.1016/j.toxlet.2008.06.672 M28 Effects of an oral subacute administration of benzo(a)pyrene on cerebral CYP1A1 expression and anxiety-related behaviour in adult mice Cao Lei 1 , Nathalie Grova 2,∗ , Claude P. Muller 2 , Henri Schroeder 1 1
URAFPA, INRA UC340, Vandoeuvre les Nancy, France, Immunology, Luxembourg City, Luxembourg
2
Institute of
Environmental pollutants such as benzo(a)pyrene (BaP) produce numerous deficits related to central deficiencies, including decreased motor activity, neuromuscular and autonomic abnormalities. While the carcinogenicity of BaP is well established, its neurotoxic effects have received less attention. Recently, some authors predicted the neurotoxic potential of polycyclic aromatic hydrocarbon particles deposited within the brain, and their subsequent adverse effects on development. Currently, the relationship between BaP, the cerebral metabolism of xenobiotics, and behaviour has received little attention. Therefore, we focused the present study on the effects of an oral subacute BaP administration (0.02, 0.2, 2, 20 and 200 mg/kg/day, 10 days) on the regional expression of CYP1A1 gene in the brain, and the anxiety-related behaviour in adult mice. The results showed that the subacute exposure to BaP induced significant increases in the CYP1A1 expression in the
hippocampus, the frontal cortex, and the temporal cortex at the two highest doses administered (20 and 200 mg/kg) whereas the lowest BaP concentrations did not. At the same doses, the animals exhibited a reduction of their anxiety level, as reflected by significant increases in the time spent in the central part of the elevated plus maze correlated with reductions in the time spent in the closed arms. Concomitantly, significant increases in the number of head dipping were observed with the same animals in the hole board apparatus. The present study suggests that there is a dose-dependent relationship between the level of BaP exposure, the cerebral metabolism of this xenobiotic, and the modulation of anxiety-related behaviour. doi:10.1016/j.toxlet.2008.06.673 M29 Time course effects of lithium administration on spatial memory acquisition and cholinergic marker expression in rats Mohammad Sharifzadeh 1,∗ , Masoomeh Pourghorban 1 , Mohammad Abdollahi 2 1
Tehran university of Medical Sciences, Tehran, Islamic Republic of Iran, 2 Pharmaceutical and Medicinal Plants Research Centers, Tehran, Islamic Republic of Iran We investigated a time course of the effects of lithium, administered systemically, on spatial memory acquisition in Morris water maze. Lithium (600 mg/L) was administered to four groups of rats in their drinking water; the first group of animals received lithium for 1 week, the second group for 2 weeks, the third group for 3 weeks, and the fourth group for 4 weeks. As controls, four groups of animals received only normal drinking water for similar periods of time. Toward the end of their lithium or water treatment, all animals were trained for four days; each day included two blocks and each block contained four trials. Test trials were conducted 48 h after completion of the lithium treatment. Lithium treatment decreased escape latency and traveled distance, but not swimming speed, compared with controls, suggesting significant spatial memory acquisition enhancement by lithium. Brain tissue sections from these animals were subjected to immunohistochemical staining analysis with anti-ChAT antibodies. Quantitative analysis showed that lithium, particularly after 4 weeks of exposure, apparently significantly increased the number and density of immunostained ChAT-containing neurons in the medial septal area compared with controls. There is also a significant correlation between the number of immunostained ChAT neurons and behavioral measures. These results suggest that systematic exposure to chronic lithium causes spatial memory acquisition improvement in rats and an apparent increase in ChAT immunostaining levels in their medial septal nuclei. doi:10.1016/j.toxlet.2008.06.674 M30 Cigarette smoke and high-fat/high-cholesterol diet induce atherogenesis in ApoE−/− -mice by different pathways Katrin Stolle 1,∗ , Sam Ansari 1 , An Berges 2 , Michael Lietz 1 , Stefan Lebrun 3 , Raymond Schleef 4 , Thomas Wallerath 1 1
PHILIP MORRIS Research Laboratories GmbH, Cologne, Germany, PHILIP MORRIS Research Laboratories bvba, Leuven, Belgium, 3 Philip Morris Products S.A., PMI Research & Development, Neuchatel, Switzerland, 4 Philip Morris USA, Richmond, VA, United States 2
Abstracts / Toxicology Letters 180S (2008) S32–S246
High-fat/high-cholesterol diet (HF) and smoking are risk factors for atherosclerosis and are typical features of a “western society life style”. In order to induce atherosclerosis in the aortic arch, apolipoproteinE-deficient (ApoE−/− ) mice were fed a HF diet or a normal chow diet for 30 days or fed a normal chow diet and exposed for 5 or 30 days to mainstream smoke (MS) from the University of Kentucky Reference Cigarette 2R4F. To elucidate the underlying mechanisms of HF- and MS-induced atherosclerosis, the gene expression pattern of the aortic arch was analyzed by DNA microarray and the following were investigated: “acute smoke effect” (5 days smoke vs. 5 days sham), “subchronic smoke effect” (30 days smoke vs. 30 days sham), “subchronic HF effect” (30 days HF diet vs. 30 days normal chow diet), and “aging effect” (30 days sham vs. 5 days sham). Hierarchical clustering and VENN analysis revealed that the different treatment groups showed mainly different sets of differentially expressed genes. Further pathway analysis (ingenuity) showed the unique and significant involvement of nine pathways in response to MS exposure for 30 days, including pathways relevant in atherogenesis, e.g., nitric oxide signaling and NRF2-mediated oxidative stress response. In response to HF diet, 14 other pathways were detected that are known to play a role in atherogenesis, e.g., NFB- and GM-CSF-signaling. In addition to these unique pathways, two pathways were shared by both treatments, i.e., PDGF-signaling and purine metabolism. These results indicate that different risk factors induce atherogenesis by different disease mechanisms. doi:10.1016/j.toxlet.2008.06.675 M31 Oxidative stress in cerebellar granule neurons exposed to methylmercury and manganese Marcelo Farina 1,∗ , Raul Bonne 1 , Iolanda ˜ 1 Campos 1 , Breno Pannia 2 , Cristina Sunol
Vendrell 1 , Francisco
1
Institut d’Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, Spain, 2 Instituto de Química, Universidad de Sao Paulo, Sao Paulo, Brazil Oxidative stress has been pointed as an important phenomenon related to metal-induced neurotoxicity. Particularly important, both methylmercury and manganese present pro-oxidative properties, which are related, at least in part, to their neurotoxic effects. However, the whole molecular mechanisms involved with methylmercury- and manganese-induced neuronal oxidative damage are still elusive. We investigated the involvement of the glutathione antioxidant system in the neurotoxicity elicited by methylmercury and manganese compounds in cultured cerebellar granule neurons. Either methylmercury or manganese II chloride induced neuronal death at 3E-7 M and 1.8E-5 M after 5 days of treatment, respectively. At time/concentrations that preceded neuronal death, manganese chloride, but not methylmercury, induced a significant increase in the neuronal glutathione levels. Ascorbic acid and lactate, but not probucol and trolox, prevented manganese-induced neuronal toxicity. Before neuronal death, methylmercury did change neither glutathione levels nor glutathione reductase activity. Conversely, a significant decrease in glutathione peroxidase was observed before neuronal death in methylmercury-exposed neurons (5 div; 300 nM). In close agreement with this observation, methylmercury increased isoprostane levels and the antioxidants ascorbic acid, trolox and probucol prevented methylmercury-induced neuronal death at 7 div. However, only probucol prevented methylmercury-induced lipid peroxidation and inhibition of glutathione peroxidase activity, rendering
S41
it a promising molecule for pharmacologic studies on methylmercury meurotoxicity. Taken together, the presented results indicate that methylmercury and manganese chloride induces neuronal oxidative damage by different pathways, where the selenoprotein glutathione peroxidase appears to be an important initial target involved in methylmercury-induced neuronal death. *Equal contribution to the work. Financed by FIS PI 06/1212. M.F. and R.B are recipients of post-doctoral and PhD fellowships from the CNPq (201362/2007-4) and USP and FAPESP (06/00001-3), Brazil. doi:10.1016/j.toxlet.2008.06.676 M32 Oxidative and immune response in experimental exposure to wooden dust A possible protective effect of the vitamin C Didi Surcel ∗ , Mariana Botoc, Mioara Butan, Anica David, Augusta Sarbu, Septimiu Toader Institute of Public Health, Cluj-Napoca, Romania Excess risk of cancer due to wood dust (WD) exposure has been suggested by epidemiologic studies, but wood dust toxicity mechanisms is a subject of discussion yet. Wooden dust—activated macrophages (AMs) become high secretory cells and release several factors, such as: interleukin-1 (IL-1), tumor necrosis factor (TNF), reactive oxygen species (ROS). Recently, there has been shown considerable interest on the vitamin C effects in immune and oxidative reactions. The aim of this study was to show the effects of vitamin C on WD-induced reactions that could be involved in the development of the precancerous conditions. An in vivo experiment was carried out on 80 Wistar rats that were divided into four groups as following: control-group; vitamin C-group; WD-group; WD + vitamin C-group. All of the groups were divided in two lots. Half of the lots were sacrificed after 1 month and another half after 6 months. In the exposed lots, WD with particles smaller than 3 m and quantity of the 5 mg/ml physiologic saline was intratracheally instilled in the rats. Vitamin C, 1 g/kg body, was administered per oral. The following parameters were assessed: (1) 3HTdR incorporation test; (2) IL-1-assay; (3) TNF-assay; (4) chemiluminiscence assay. Our results point out the following findings: wooden dust interferes with oxidative and immune reactions; AMs play a pivotal role by releasing high levels of IL-1, TNF, ROS; vitamin C may have a protective effect in wooden dust exposure by its immunomodulatory and antioxidant effects. doi:10.1016/j.toxlet.2008.06.677 M33 Benzo(a)pyrene induced apoptosis in MCF-7 cells Marjo Tampio 1,∗ , Piia Markkanen 2 , Maija-Riitta Hirvonen 2 , Kirsi H. Vähäkangas 1 1 University on Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland, 2 National Public Health Institute, Department of Environmental Health, Kuopio, Finland
p53 tumor suppressor protein is important in the regulation of cell cycle. DNA repair and apoptosis all of which are needed to maintain the genome intact. p53 stability and activity are mainly regulated by post-translational modifications. The most studied p53 phosphorylations, serines 15 and 20, are known to prevent degradation of p53 by impairing the interaction between p53 and
Abstracts / Toxicology Letters 180S (2008) S32–S246
as an agonist of the constitutive androstane receptor (CAR) and may also have some agonistic activity at the Ah receptor. doi:10.1016/j.toxlet.2008.06.671 M27 Investigation of the brain distribution of the neuronal cytochrome oxidase activity in adult mice submitted to a subacute benzo(a)pyrene administration Julie Peiffer 1 , Catherine Strazielle 2 , Henri Schroeder 1,∗ 1
URAFPA, INRA UC340, Vandoeuvre les Nancy, France, U724, Vandoeuvre les Nancy, France
2
INSERM
A 10-day subacute administration of benzo(a)pyrene (BaP) has been shown to induce specific behavioural alterations including spatial memory and anxiety, in relation with significant variations of the NR1 and NR2a NMDA receptor subunit expression located in the hippocampus, the temporal cortex, and the frontal cortex. In order to precise the sensitivity of cerebral areas to BaP, the regional distribution of cytochrome oxidase (CO) activity, a mitochondrial enzyme marker for neuronal activation, was investigated in mice treated with BaP. Groups of six mice each were daily injected i.p. with BaP (0.02, 0.2, 2, 20, 200 mg/kg/day) for 10 days. At the end of the period of treatment, the mice were sacrificed, and brains were removed and deep frozen. Thus, frontal sections of 20-m thick were collected from each brain. The CO activity was revealed by histochemistry using the 3–3 diaminobenzidine as substrate, and measured in 68 structures using an image analysis densitometer. The results showed that BaP induced significant increases in CO activity in 43 of the 68 brain areas studied. Over the range of doses injected, the CO activity was significantly increased in mainly limbic structures such as the amygdala, the hippocampus, the lateral septum, some specific hypothalamus and thalamus nuclei, and various cortical areas including the entorhinal, perirhinal, piriform, and cingular cortices. No significant variation was observed in both motor and sensory areas. These findings suggest that BaP induced specific alterations of the cerebral activity in regions that highly correlated with the behavioural disorders previously observed. doi:10.1016/j.toxlet.2008.06.672 M28 Effects of an oral subacute administration of benzo(a)pyrene on cerebral CYP1A1 expression and anxiety-related behaviour in adult mice Cao Lei 1 , Nathalie Grova 2,∗ , Claude P. Muller 2 , Henri Schroeder 1 1
URAFPA, INRA UC340, Vandoeuvre les Nancy, France, Immunology, Luxembourg City, Luxembourg
2
Institute of
Environmental pollutants such as benzo(a)pyrene (BaP) produce numerous deficits related to central deficiencies, including decreased motor activity, neuromuscular and autonomic abnormalities. While the carcinogenicity of BaP is well established, its neurotoxic effects have received less attention. Recently, some authors predicted the neurotoxic potential of polycyclic aromatic hydrocarbon particles deposited within the brain, and their subsequent adverse effects on development. Currently, the relationship between BaP, the cerebral metabolism of xenobiotics, and behaviour has received little attention. Therefore, we focused the present study on the effects of an oral subacute BaP administration (0.02, 0.2, 2, 20 and 200 mg/kg/day, 10 days) on the regional expression of CYP1A1 gene in the brain, and the anxiety-related behaviour in adult mice. The results showed that the subacute exposure to BaP induced significant increases in the CYP1A1 expression in the
hippocampus, the frontal cortex, and the temporal cortex at the two highest doses administered (20 and 200 mg/kg) whereas the lowest BaP concentrations did not. At the same doses, the animals exhibited a reduction of their anxiety level, as reflected by significant increases in the time spent in the central part of the elevated plus maze correlated with reductions in the time spent in the closed arms. Concomitantly, significant increases in the number of head dipping were observed with the same animals in the hole board apparatus. The present study suggests that there is a dose-dependent relationship between the level of BaP exposure, the cerebral metabolism of this xenobiotic, and the modulation of anxiety-related behaviour. doi:10.1016/j.toxlet.2008.06.673 M29 Time course effects of lithium administration on spatial memory acquisition and cholinergic marker expression in rats Mohammad Sharifzadeh 1,∗ , Masoomeh Pourghorban 1 , Mohammad Abdollahi 2 1
Tehran university of Medical Sciences, Tehran, Islamic Republic of Iran, 2 Pharmaceutical and Medicinal Plants Research Centers, Tehran, Islamic Republic of Iran We investigated a time course of the effects of lithium, administered systemically, on spatial memory acquisition in Morris water maze. Lithium (600 mg/L) was administered to four groups of rats in their drinking water; the first group of animals received lithium for 1 week, the second group for 2 weeks, the third group for 3 weeks, and the fourth group for 4 weeks. As controls, four groups of animals received only normal drinking water for similar periods of time. Toward the end of their lithium or water treatment, all animals were trained for four days; each day included two blocks and each block contained four trials. Test trials were conducted 48 h after completion of the lithium treatment. Lithium treatment decreased escape latency and traveled distance, but not swimming speed, compared with controls, suggesting significant spatial memory acquisition enhancement by lithium. Brain tissue sections from these animals were subjected to immunohistochemical staining analysis with anti-ChAT antibodies. Quantitative analysis showed that lithium, particularly after 4 weeks of exposure, apparently significantly increased the number and density of immunostained ChAT-containing neurons in the medial septal area compared with controls. There is also a significant correlation between the number of immunostained ChAT neurons and behavioral measures. These results suggest that systematic exposure to chronic lithium causes spatial memory acquisition improvement in rats and an apparent increase in ChAT immunostaining levels in their medial septal nuclei. doi:10.1016/j.toxlet.2008.06.674 M30 Cigarette smoke and high-fat/high-cholesterol diet induce atherogenesis in ApoE−/− -mice by different pathways Katrin Stolle 1,∗ , Sam Ansari 1 , An Berges 2 , Michael Lietz 1 , Stefan Lebrun 3 , Raymond Schleef 4 , Thomas Wallerath 1 1
PHILIP MORRIS Research Laboratories GmbH, Cologne, Germany, PHILIP MORRIS Research Laboratories bvba, Leuven, Belgium, 3 Philip Morris Products S.A., PMI Research & Development, Neuchatel, Switzerland, 4 Philip Morris USA, Richmond, VA, United States 2
Abstracts / Toxicology Letters 180S (2008) S32–S246
High-fat/high-cholesterol diet (HF) and smoking are risk factors for atherosclerosis and are typical features of a “western society life style”. In order to induce atherosclerosis in the aortic arch, apolipoproteinE-deficient (ApoE−/− ) mice were fed a HF diet or a normal chow diet for 30 days or fed a normal chow diet and exposed for 5 or 30 days to mainstream smoke (MS) from the University of Kentucky Reference Cigarette 2R4F. To elucidate the underlying mechanisms of HF- and MS-induced atherosclerosis, the gene expression pattern of the aortic arch was analyzed by DNA microarray and the following were investigated: “acute smoke effect” (5 days smoke vs. 5 days sham), “subchronic smoke effect” (30 days smoke vs. 30 days sham), “subchronic HF effect” (30 days HF diet vs. 30 days normal chow diet), and “aging effect” (30 days sham vs. 5 days sham). Hierarchical clustering and VENN analysis revealed that the different treatment groups showed mainly different sets of differentially expressed genes. Further pathway analysis (ingenuity) showed the unique and significant involvement of nine pathways in response to MS exposure for 30 days, including pathways relevant in atherogenesis, e.g., nitric oxide signaling and NRF2-mediated oxidative stress response. In response to HF diet, 14 other pathways were detected that are known to play a role in atherogenesis, e.g., NFB- and GM-CSF-signaling. In addition to these unique pathways, two pathways were shared by both treatments, i.e., PDGF-signaling and purine metabolism. These results indicate that different risk factors induce atherogenesis by different disease mechanisms. doi:10.1016/j.toxlet.2008.06.675 M31 Oxidative stress in cerebellar granule neurons exposed to methylmercury and manganese Marcelo Farina 1,∗ , Raul Bonne 1 , Iolanda ˜ 1 Campos 1 , Breno Pannia 2 , Cristina Sunol
Vendrell 1 , Francisco
1
Institut d’Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, Barcelona, Spain, 2 Instituto de Química, Universidad de Sao Paulo, Sao Paulo, Brazil Oxidative stress has been pointed as an important phenomenon related to metal-induced neurotoxicity. Particularly important, both methylmercury and manganese present pro-oxidative properties, which are related, at least in part, to their neurotoxic effects. However, the whole molecular mechanisms involved with methylmercury- and manganese-induced neuronal oxidative damage are still elusive. We investigated the involvement of the glutathione antioxidant system in the neurotoxicity elicited by methylmercury and manganese compounds in cultured cerebellar granule neurons. Either methylmercury or manganese II chloride induced neuronal death at 3E-7 M and 1.8E-5 M after 5 days of treatment, respectively. At time/concentrations that preceded neuronal death, manganese chloride, but not methylmercury, induced a significant increase in the neuronal glutathione levels. Ascorbic acid and lactate, but not probucol and trolox, prevented manganese-induced neuronal toxicity. Before neuronal death, methylmercury did change neither glutathione levels nor glutathione reductase activity. Conversely, a significant decrease in glutathione peroxidase was observed before neuronal death in methylmercury-exposed neurons (5 div; 300 nM). In close agreement with this observation, methylmercury increased isoprostane levels and the antioxidants ascorbic acid, trolox and probucol prevented methylmercury-induced neuronal death at 7 div. However, only probucol prevented methylmercury-induced lipid peroxidation and inhibition of glutathione peroxidase activity, rendering
S41
it a promising molecule for pharmacologic studies on methylmercury meurotoxicity. Taken together, the presented results indicate that methylmercury and manganese chloride induces neuronal oxidative damage by different pathways, where the selenoprotein glutathione peroxidase appears to be an important initial target involved in methylmercury-induced neuronal death. *Equal contribution to the work. Financed by FIS PI 06/1212. M.F. and R.B are recipients of post-doctoral and PhD fellowships from the CNPq (201362/2007-4) and USP and FAPESP (06/00001-3), Brazil. doi:10.1016/j.toxlet.2008.06.676 M32 Oxidative and immune response in experimental exposure to wooden dust A possible protective effect of the vitamin C Didi Surcel ∗ , Mariana Botoc, Mioara Butan, Anica David, Augusta Sarbu, Septimiu Toader Institute of Public Health, Cluj-Napoca, Romania Excess risk of cancer due to wood dust (WD) exposure has been suggested by epidemiologic studies, but wood dust toxicity mechanisms is a subject of discussion yet. Wooden dust—activated macrophages (AMs) become high secretory cells and release several factors, such as: interleukin-1 (IL-1), tumor necrosis factor (TNF), reactive oxygen species (ROS). Recently, there has been shown considerable interest on the vitamin C effects in immune and oxidative reactions. The aim of this study was to show the effects of vitamin C on WD-induced reactions that could be involved in the development of the precancerous conditions. An in vivo experiment was carried out on 80 Wistar rats that were divided into four groups as following: control-group; vitamin C-group; WD-group; WD + vitamin C-group. All of the groups were divided in two lots. Half of the lots were sacrificed after 1 month and another half after 6 months. In the exposed lots, WD with particles smaller than 3 m and quantity of the 5 mg/ml physiologic saline was intratracheally instilled in the rats. Vitamin C, 1 g/kg body, was administered per oral. The following parameters were assessed: (1) 3HTdR incorporation test; (2) IL-1-assay; (3) TNF-assay; (4) chemiluminiscence assay. Our results point out the following findings: wooden dust interferes with oxidative and immune reactions; AMs play a pivotal role by releasing high levels of IL-1, TNF, ROS; vitamin C may have a protective effect in wooden dust exposure by its immunomodulatory and antioxidant effects. doi:10.1016/j.toxlet.2008.06.677 M33 Benzo(a)pyrene induced apoptosis in MCF-7 cells Marjo Tampio 1,∗ , Piia Markkanen 2 , Maija-Riitta Hirvonen 2 , Kirsi H. Vähäkangas 1 1 University on Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland, 2 National Public Health Institute, Department of Environmental Health, Kuopio, Finland
p53 tumor suppressor protein is important in the regulation of cell cycle. DNA repair and apoptosis all of which are needed to maintain the genome intact. p53 stability and activity are mainly regulated by post-translational modifications. The most studied p53 phosphorylations, serines 15 and 20, are known to prevent degradation of p53 by impairing the interaction between p53 and