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Kobets T, Iatropoulos MJ, Duan JD, Brunnemann KD, Iacobas DA, Iacobas S, Vock E, Deschl U, Williams GM. Expression of Genes Encoding for Xenobiotic Metabolism After Exposure to Dialkylnitrosamines in the Chicken Egg Genotoxicity Alternative Model. Toxicol Sci 2019; 166:82-96. [PMID: 30102407 DOI: 10.1093/toxsci/kfy197] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The Chicken Egg Genotoxicity Assay (CEGA) demonstrated responsiveness to various DNA-reactive chemicals requiring metabolic activation, which implies broad bioactivation capability. To assess potential metabolic competence, expression profiles of metabolic genes in the embryo-chicken fetal liver were determined using microarray technology. Fertilized chicken eggs were injected under the CEGA protocol with vehicle (deionized water [DW]), the activation-dependent carcinogens, diethylnitrosamine (DEN), and N-nitrosodiethanolamine (NDELA) at doses producing no effect on survival. Previously in CEGA, DEN produced DNA damage, whereas NDELA did not. Expressions of 463 genes known to encode for phase I and II of endo- and xenobiotic metabolism were detected on the array. DW did not affect the expression of the selected genes, deregulating less than 1% of them. In contrast, DEN at 2 mg/egg and NDELA at 4 mg/egg produced significant transcriptomic alterations, up-regulating up to 41% and down-regulating over 31% of studied genes. Both nitrosamines modulated the majority of the genes in a similar manner, sharing 64 up-regulated and 93 down-regulated genes with respect to control group, indicating similarity in the regulation of their metabolism by avian liver. Differences in gene expression between DEN and NDELA were documented for several phase I CYP 450 genes that are responsible for nitrosamine biotransformation, as well as for phase II genes that regulate detoxication reactions. These findings could underlie the difference in genotoxicity of DEN and NDELA in CEGA. In conclusion, the analysis of gene expression profiles in embryo-chicken fetal liver dosed with dialkylnitrosamines demonstrated that avian species possess a complex array of inducible genes coding for biotransformation.
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Affiliation(s)
- Tetyana Kobets
- Department of Pthology, New York Medical College, Valhalla, New York 10595
| | | | - Jiandong D Duan
- Department of Pthology, New York Medical College, Valhalla, New York 10595
| | - Klaus D Brunnemann
- Department of Pthology, New York Medical College, Valhalla, New York 10595
| | - Dumitru A Iacobas
- Center for Computational Systems Biology, Prairie View A&M University, Prairie View, Texas 77446
| | - Sanda Iacobas
- Department of Pthology, New York Medical College, Valhalla, New York 10595
| | - Esther Vock
- Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach an der Riss, Germany 88397
| | - Ulrich Deschl
- Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach an der Riss, Germany 88397
| | - Gary M Williams
- Department of Pthology, New York Medical College, Valhalla, New York 10595
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Becker RA, Dellarco V, Seed J, Kronenberg JM, Meek B, Foreman J, Palermo C, Kirman C, Linkov I, Schoeny R, Dourson M, Pottenger LH, Manibusan MK. Quantitative weight of evidence to assess confidence in potential modes of action. Regul Toxicol Pharmacol 2017; 86:205-220. [DOI: 10.1016/j.yrtph.2017.02.017] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2016] [Revised: 02/17/2017] [Accepted: 02/18/2017] [Indexed: 12/31/2022]
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3
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Nakagawa K, Hosoe K, Hidaka T, Nabae K, Kawabe M, Kitano M. Inhibition by licorice flavonoid oil of glutathione S-transferase–positive foci in the medium-term rat hepatocarcinogenesis bioassay. Nutr Res 2010; 30:74-81. [DOI: 10.1016/j.nutres.2009.12.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2009] [Revised: 12/05/2009] [Accepted: 12/17/2009] [Indexed: 11/27/2022]
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Klaunig JE, Babich MA, Baetcke KP, Cook JC, Corton JC, David RM, DeLuca JG, Lai DY, McKee RH, Peters JM, Roberts RA, Fenner-Crisp PA. PPARα Agonist-Induced Rodent Tumors: Modes of Action and Human Relevance. Crit Rev Toxicol 2008; 33:655-780. [PMID: 14727734 DOI: 10.1080/713608372] [Citation(s) in RCA: 449] [Impact Index Per Article: 26.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Widely varied chemicals--including certain herbicides, plasticizers, drugs, and natural products--induce peroxisome proliferation in rodent liver and other tissues. This phenomenon is characterized by increases in the volume density and fatty acid oxidation of these organelles, which contain hydrogen peroxide and fatty acid oxidation systems important in lipid metabolism. Research showing that some peroxisome proliferating chemicals are nongenotoxic animal carcinogens stimulated interest in developing mode of action (MOA) information to understand and explain the human relevance of animal tumors associated with these chemicals. Studies have demonstrated that a nuclear hormone receptor implicated in energy homeostasis, designated peroxisome proliferator-activated receptor alpha (PPARalpha), is an obligatory factor in peroxisome proliferation in rodent hepatocytes. This report provides an in-depth analysis of the state of the science on several topics critical to evaluating the relationship between the MOA for PPARalpha agonists and the human relevance of related animal tumors. Topics include a review of existing tumor bioassay data, data from animal and human sources relating to the MOA for PPARalpha agonists in several different tissues, and case studies on the potential human relevance of the animal MOA data. The summary of existing bioassay data discloses substantial species differences in response to peroxisome proliferators in vivo, with rodents more responsive than primates. Among the rat and mouse strains tested, both males and females develop tumors in response to exposure to a wide range of chemicals including DEHP and other phthalates, chlorinated paraffins, chlorinated solvents such as trichloroethylene and perchloroethylene, and certain pesticides and hypolipidemic pharmaceuticals. MOA data from three different rodent tissues--rat and mouse liver, rat pancreas, and rat testis--lead to several different postulated MOAs, some beginning with PPARalpha activation as a causal first step. For example, studies in rodent liver identified seven "key events," including three "causal events"--activation of PPARalpha, perturbation of cell proliferation and apoptosis, and selective clonal expansion--and a series of associative events involving peroxisome proliferation, hepatocyte oxidative stress, and Kupffer-cell-mediated events. Similar in-depth analysis for rat Leydig-cell tumors (LCTs) posits one MOA that begins with PPARalpha activation in the liver, but two possible pathways, one secondary to liver induction and the other direct inhibition of testicular testosterone biosynthesis. For this tumor, both proposed pathways involve changes in the metabolism and quantity of related hormones and hormone precursors. Key events in the postulated MOA for the third tumor type, pancreatic acinar-cell tumors (PACTs) in rats, also begin with PPARalpha activation in the liver, followed by changes in bile synthesis and composition. Using the new human relevance framework (HRF) (see companion article), case studies involving PPARalpha-related tumors in each of these three tissues produced a range of outcomes, depending partly on the quality and quantity of MOA data available from laboratory animals and related information from human data sources.
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Affiliation(s)
- James E Klaunig
- Indiana University School of Medicine, Indianapolis, IN, USA
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5
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Guo YT, Leng XS, Li T, Zhao JM, Lin XH. Peroxisome proliferator-activated receptor γ ligands suppress liver carcinogenesis induced by diethylnitrosamine in rats. World J Gastroenterol 2004; 10:3419-23. [PMID: 15526359 PMCID: PMC4576221 DOI: 10.3748/wjg.v10.i23.3419] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Peroxisome proliferator-activated receptor γ (PPARγ ) is known to regulate growth arrest and terminal differentiation of adipocytes and is used clinically as a new class of antidiabetic drugs. Recently, several studies have reported that treatment of cancer cells with PPARγ ligands could induce cell differentiation and apoptosis, suggesting a potential application as chemopreventive agents against carcinogenesis. In the present study, 3 different kinds of PPARγ ligands were subjected to the experiments to confirm their suppressive effects on liver carcinogenesis.
METHODS: Three PPARγ ligands, pioglitazone (Pio) (200 ppm), rosiglitazone (Rosi) (200 ppm), and troglitazone (Tro) (1000 ppm) were investigated on the induction of the placental form of rat glutathione S-transferase (rGST P) positive foci, a precancerous lesion of the liver,and liver cancer formation using a diethylnitrosamine-induced liver cancer model in Wistar rats, and dose dependency of a PPARγ ligand was also examined.
RESULTS: PPARγ ligands reduced the formation of rGST P-positive foci by diethylnitrosamine and induction of liver cancers was also markedly suppressed by a continuous feeding of Pio at 200 ppm.
CONCLUSION: PPARγ ligands are potential chemopreventive agents for liver carcinogenesis.
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Affiliation(s)
- Yan-Tong Guo
- Department of General Surgery, Beijing Jishuitan Hospital, the Forth Clinical Medical College of Peking University, Beijing 100035, China.
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Michel C, Desdouets C, Sacre-Salem B, Gautier JC, Roberts R, Boitier E. Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver. THE AMERICAN JOURNAL OF PATHOLOGY 2004; 163:2191-9. [PMID: 14633594 PMCID: PMC1892366 DOI: 10.1016/s0002-9440(10)63577-8] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.
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Affiliation(s)
- Cécile Michel
- Department of Drug Safety Evaluation, Aventis Pharma, Vitry-sur-Seine, France
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Kaji I, Tatsuta M, Iishi H, Baba M, Inoue A, Kasugai H. Inhibition by d-limonene of experimental hepatocarcinogenesis in Sprague-Dawley rats does not involve p21(ras) plasma membrane association. Int J Cancer 2001; 93:441-4. [PMID: 11433412 DOI: 10.1002/ijc.1353] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The effects of d-limonene on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and on membrane-associated p21(ras) and labeling and apoptotic indices of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of experimental week 9, they received chow pellets containing 1% or 2% limonene. The preneoplastic and neoplastic liver lesions (cellular alteration foci, neoplastic nodules and hepatocellular carcinomas), and hepatic foci staining positive for glutathione-S-transferase, placental type (GST-P) were examined microscopically and histochemically. At week 16, quantitative histologic analysis showed that oral administration of 1% or 2% limonene resulted in significant reductions in the number and mean area of GST-P-positive hepatic foci and the number of cellular alteration foci, neoplastic nodules and hepatocellular carcinomas. Limonene, at both doses, also caused significant decreases in the labeling indices and significant increases in the apoptotic indices of cellular alteration foci, neoplastic nodules, hepatocellular carcinomas and adjacent liver. However, limonene, at both doses, had no significant influence on the production of membrane-associated p21(ras) in the visible liver white nodules. These findings indicate that limonene inhibits hepatocarcinogenesis and suggest that this effect may be clearly related to its effect in inhibiting cell proliferation and in enhancing apoptosis, but not through ras oncoprotein plasma membrane association.
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Affiliation(s)
- I Kaji
- Department of Gastroenterology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.
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8
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Ledda-Columbano GM, Pibiri M, Perra A, Piga R, Loi R, Dore M, Shinozuka H, Columbano A. Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes. Cancer Lett 2000; 151:153-9. [PMID: 10738109 DOI: 10.1016/s0304-3835(99)00407-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.
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Affiliation(s)
- G M Ledda-Columbano
- Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Via Porcell 4, 09124, Cagliari, Italy.
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9
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Ledda-Columbano GM, Perra A, Piga R, Pibiri M, Loi R, Shinozuka H, Columbano A. Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions. Carcinogenesis 1999; 20:2299-304. [PMID: 10590223 DOI: 10.1093/carcin/20.12.2299] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.
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Affiliation(s)
- G M Ledda-Columbano
- Dipartimento di Tossicologia, Sezione di Oncologia e Patologia Molecolare, Via Porcell 4, 09124 Cagliari, Italy.
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10
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Kim DJ, Lee KK, Hong JT. Differential effects of nongenotoxic and genotoxic carcinogens on the preneoplastic lesions in the rat liver. Arch Pharm Res 1998; 21:363-9. [PMID: 9875460 DOI: 10.1007/bf02974627] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Glutathione S-transferase placental form (GST-P) positive foci development and its expression in liver exposed by nongenotoxic carcinogens phenobarbital (PB) and clofibrate (CF), and genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) were investigated as a measure of carcinogenic potential of these chemicals. Male F344 rats were initially given a single intraperitioneal injection of diethylnitrosamine (200 mg/kg), and 2 weeks later, animals were fed diets containing 0.03% IQ or 0.5% CF or 0.05% PB or basal diet as a control for 6 weeks. All rats were subjected to two-thirds partial hepatectomy (PH) at week 3. Sequential sacrifice of rats was performed at 8 weeks or 52 weeks, and liver tissues were examined for immunohistochemical staining of GST-P positive foci. The numbers (No./cm2) and areas (mm2/cm2) of GST-P positive foci were increased by IQ or PB, but were decreased by CF compare to the control. Consistent with the development of GST-P positive foci, a time-related increase in the expression of GST-P mRNA was found in the rats treated with IQ, whereas CF decreased it. The incidence of hepatocellular carcinoma at 52 weeks was increased by all three chemicals. These results show that PB and IQ induced GST-P positive foci, but the peroxisome proliferator CF did not, which suggest that the prediction of carcinogenic potency based on the development of prenoplastic foci may cause false negative in a particular category compounds like peroxisome proliferators.
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Affiliation(s)
- D J Kim
- Department of Pathology, National Institute of Toxicology Research, Korea Food and Drug Administration, Seoul
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11
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Thullberg M, Grasl-Kraupp B, Högberg J, Garberg P. Changes in liver fatty acid-binding protein in rat enzyme-altered foci. Cancer Lett 1998; 128:1-10. [PMID: 9652787 DOI: 10.1016/s0304-3835(98)00040-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The level of liver fatty acid-binding protein (L-FABP) was analyzed in enzyme-altered foci (EAF) positive for GST-P, or after classification of foci into different subclasses by haematoxylin and eosin staining. Rats were treated with either an initiating single dose of diethylnitrosamine (DEN) followed by no treatment, treatment with phenobarbital, PCB, nafenopin or repeated injections of DEN, or alternatively non-treated or treated with nafenopin alone. Changes in the level of L-FABP were detected in the majority of EAF and both L-FABP-positive and -negative foci were seen. However, in rats initiated with DEN, EAF were almost exclusively L-FABP-negative. The fraction of L-FABP-negative foci increased with increasing foci size, while the time of treatment or the dose of the promoter did not seem to have any effect. It was also found that treatment with DEN gave a higher fraction of L-FABP-negative foci as compared to treatment with phenobarbital or PCB, indicating a specific effect of DEN. These data together with previously published findings suggest that L-FABP expression in EAF is determined by the initiating carcinogenic regimen and that it might be possible to use the expression of L-FABP in tumours to differentiate initiating chemicals.
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Affiliation(s)
- M Thullberg
- Department of Toxicology and Chemistry, National Institute for Working Life, Solna, Sweden
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12
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13
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Affiliation(s)
- M S Rao
- Department of Pathology, Northwestern University, Medical School, Chicago, Illinois 60611, USA
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14
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Ito S, Tateno C, Tuda M, Yoshitake A. Immunohistochemical demonstration of the gap junctional protein connexin 32 and proliferating cell nuclear antigen in glutathione S-transferase placental form-negative lesions of rat liver induced by diethylnitrosamine and clofibrate. Toxicol Pathol 1996; 24:690-5. [PMID: 8994295 DOI: 10.1177/019262339602400603] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The distributions of a gap junctional protein, connexin 32 (cx 32), and proliferating cell nuclear antigen (PCNA) were examined immunohistochemically in glutathione S-transferase placental form (GST-P)-negative foci, induced in rat liver by initiation with diethylnitrosamine (DEN, 200 mg/kg) followed by promotion with clofibrate (1% in diet) in an in vivo medium-term assay system for hepatocarcinogenesis. The results were compared to those in GST-P-positive foci induced by DEN alone. The treatment with clofibrate caused the appearance of GST-P-negative foci, increased in size as compared to GST-P-positive foci in the same liver or induced by the DEN alone. The proportion of PCNA-positive hepatocytes in GST-P-negative foci was significantly higher than in the surrounding parenchyma, indicating increased cell proliferation. The numbers of cx 32-positive spots per hepatocyte in GST-P-negative foci were clearly decreased, reaching 65.4% at week 20 and 51.8% at week 30 of values for surrounding normal hepatocytes. In GST-P-positive foci induced by DEN, only a slight decrease (80%) was observed at week 8. These findings show that a positive association between the sustained inhibition of gap junctional intercellular communication and increased cell proliferation of GST-P-negative foci in Fischer-344 male rats induced with DEN and promoted with clofibrate.
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Affiliation(s)
- S Ito
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan
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15
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Huber WW, Grasl-Kraupp B, Schulte-Hermann R. Hepatocarcinogenic potential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk. Crit Rev Toxicol 1996; 26:365-481. [PMID: 8817083 DOI: 10.3109/10408449609048302] [Citation(s) in RCA: 184] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The plasticizer di(2-ethylhexyl) phthalate (DEHP), to which humans are extensively exposed, was found to be hepatocarcinogenic in rats and mice. DEHP is potentially set free from objects made of synthetic materials (e.g., those used in medicine). Chronically, the greatest amounts are transferred to persons undergoing hemodialysis (up to 3.1 mg/kg b.w. per day) who would thus be considered the individuals most endangered by tumorigenesis. Although toxicokinetics seem to play a certain unclear role in the course of DEHP-related toxicity, toxicodynamic factors appear more decisive. DEHP is a representative of "peroxisome proliferators" (PP), a distinct group of substances that, in rodents, do not only induce peroxisomes but also specific enzymes in other organelles, organ growth, and DNA synthesis. The cluster of the characteristic effects of PP is generally, although perhaps not quite appropriately summarized as "peroxisome proliferation," and is strongest in the liver. The lowest observed effect level (LOEL) and the no observed effect level (NOEL) of peroxisome proliferation in the rat, as determined by the induction of specific enzymes (peroxisomal beta-oxidation, carnitine-acetyl-transferase, cytochrome P-452), DNA synthesis, and hepatomegaly, may be assumed as 50 and 25 mg/kg b.w. per day, respectively. DEHP and other carcinogenic PP are neither genotoxic nor tumor initiators, but they appear to be tumor promoters, also implicating a threshold level for the carcinogenic effect. Although a causal relationship between a particular effect of peroxisome proliferation and hepatocarcinogenesis is as yet unknown, peroxisome proliferation as a whole phenomenon appears to be associated with the potential of tumor induction, as shown by comparison of the relative strength of individual PP and by comparison of species and organ specificities. Likewise, LOEL and NOEL of rodent carcinogenesis, that is, 300 and 50 to 100 mg/kg b.w. per day, respectively, are above but not too far from the corresponding values for the investigated parameters of peroxisome proliferation. Thus, with respect to dose alone, worst-case exposure in hemodialysis patients is at least 16-fold below the LOEL of any characterized PP-specific effect of DEHP and approximately 100-fold below that of DEHP-related tumorigenesis. Also, primates are less responsive to PP than rats with respect to the investigated biochemical and morphological parameters. If this lower primate responsiveness is extrapolated to estimate carcinogenicity in humans, we might thus arrive at an even larger safety margin than when based on exposure alone. Doses of PP hypolipidemics that had clearly induced several indicators of peroxisome proliferation in rats did not cause any clear-cut enhancements in the peroxisomes of patients, even though most of these hypolipidemics were considerably stronger PP than DEHP. Thus, an actual threat to humans by DEHP seems rather unlikely. Accordingly, hepatocarcinogenesis was neither enhanced in workers exposed to DEHP nor in patients treated with hypolipidemics.
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Affiliation(s)
- W W Huber
- Institut für Tumorbiologie und Krebsforschung, University of Vienna, Austria
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16
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Tsuda H, Asamoto M, Baba-Toriyama H, Iwahori Y, Hori T, Kim DJ, Tsuchiya T, Mutai M, Yamasaki H. Clofibrate-induced neoplastic development in the rat liver is associated with decreased connexin 32 expression but not with a co-ordinated shift in expression of marker enzymes. Toxicol Lett 1995; 82-83:693-9. [PMID: 8597128 DOI: 10.1016/0378-4274(95)03587-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Altered enzyme phenotype and expression of connexin 32 (Cx32), a gap junction protein were studied during the development of rat liver tumors induced by the non-genotoxic carcinogen, clofibrate. (1) In contrast to previous findings for nitrosamine-induced lesions, preneoplastic enzyme-altered foci (EAF) and neoplastic nodules (NN) lacked any clear association with degree of altered enzyme expression because of an almost complete negativity for GST-P and GGT. (2) Immunohistochemically demonstrated Cx32 spots on the hepatocyte membranes showed a clear decrease in clofibrate-induced lesions. (3) Naturally occurring EAF demonstrating GST-P and/or GGT positivity did not show a significant decrease of Cx32 counts suggesting a reversible nature. Therefore, the Cx32 decrease appears closely linked to progression of hepatocarcinogenesis irrespective of the enzyme phenotype of neoplastic focal lesions and the carcinogens used for their induction.
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Affiliation(s)
- H Tsuda
- Chemotherapy Division, National Cancer Center Research Institute, Tokyo, Japan
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17
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Mehta R. The potential for the use of cell proliferation and oncogene expression as intermediate markers during liver carcinogenesis. Cancer Lett 1995; 93:85-102. [PMID: 7600546 DOI: 10.1016/0304-3835(95)03790-4] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Intense research using animal models has indicated that chemically-induced rat liver cancer proceeds through multiple, distinct stages that can be characterised morphologically and biochemically. Primary human liver cancer, with hepatitis B and other environmental factors such as poor nutrition and food contaminating mycotoxins as contributing etiological factors, is one of the major causes of cancer deaths in African, Asian and some Western countries. Recent advances in surgical and diagnostic techniques have also allowed the identification of potential morphological precursors of primary human liver cancer, and suggested a model consistent with the concepts of initiation--promotion--progression as in the rat. The expression of proliferating cell nuclear antigen (PCNA), silver-staining nucleolar organiser regions (AgNOR), oncogenes and the tumor suppressor gene p53 in preneoplastic and neoplastic lesions of rat and human livers is presently reviewed. This undertaking is an attempt to evaluate whether the current knowledge regarding molecular mechanisms of carcinogenesis is sufficient to permit the use of these molecular parameters as 'intermediate' markers in studies of risk assessment and cancer prevention, without having to resort to tumor appearance as an end-point.
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Affiliation(s)
- R Mehta
- Toxicology Research Division, Health Protection Branch, Health Canada, Ottawa, Ontario
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Foliot A, Beaune P. Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters. Biochem Pharmacol 1994; 48:293-300. [PMID: 8053925 DOI: 10.1016/0006-2952(94)90100-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The effects of microsomal enzyme inducers on glutathione S-transferase (GST) isoenzymes were studied in livers of rats and hamsters using three hypolipidemic drugs of the peroxisome proliferator type and the two model substances phenobarbital (PB) and 3-methylcholanthrene (MC). The effects were investigated by immunoblot analysis of the various GST subunits using polyclonal antibodies directed to rat subunits 1-4. In untreated animals the subunit composition was different, with hamsters having a much higher content of class mu isoenzymes. Administration of all three hypolipidemic drugs reduced the protein concentration of both alpha and mu class GSTs in rats but reduced only class mu subunits in hamsters. This reduction was in good agreement with the decreased activity observed with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB) in both species. As expected, PB and MC increased GST activity together with the concentration of subunits 1 and 3 in rats. In hamsters, PB significantly increased subunit 1 and slightly reduced subunits 3 and 4, although this decrease was not significant. Total GST, measured with CDNB, was reduced by 17%. In contrast, MC slightly decreased subunit 1 and markedly raised subunits 3 and 4, resulting in a net increase in total GST activity. All drugs increased relative liver weight, microsomal protein concentration and total P450 in both species; in contrast, total cytosolic proteins were raised by all drugs in rats but not in hamsters, except for MC. The results obtained in these two species show that GST activity is not always increased by microsomal enzyme inducers. The response may depend in part on isoenzyme profile, and varies with the subunit considered.
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Affiliation(s)
- A Foliot
- INSERM U 75, Université René Descartes, CHU Necker, Paris, France
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Bentley P, Calder I, Elcombe C, Grasso P, Stringer D, Wiegand HJ. Hepatic peroxisome proliferation in rodents and its significance for humans. Food Chem Toxicol 1993; 31:857-907. [PMID: 8258416 DOI: 10.1016/0278-6915(93)90225-n] [Citation(s) in RCA: 185] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Peroxisomes are subcellular organelles found in all eukaryotic cells. In the liver they are usually round and measure about 0.5-1.0 microns; in rodents they contain a prominent crystalloid core, but this may be absent in newly formed rodent peroxisomes as well as in human peroxisomes. A major role of the peroxisomes is the breakdown of long-chain fatty acids, thereby complementing mitochondrial fatty-acid metabolism. Many chemicals are known to increase the number of peroxisomes in rat and mouse hepatocytes. This peroxisome proliferation is accompanied by replicative DNA synthesis and liver growth. No clear structure-activity relationships are apparent. Many of these peroxisome proliferators contain acid functions that can modulate fatty acid metabolism. Two mechanisms have been proposed for the induction of peroxisome proliferation. One is based on the existence of one or several specific cytosolic receptors that bind the peroxisome proliferator, facilitating its translocation to the cell nucleus and the activation of the expression of specific genes. The second, perhaps more general, hypothesis involves chemically mediated perturbation of lipid metabolism. These two hypotheses are not mutually exclusive. Many peroxisome proliferators have been shown to induce hepatocellular tumours, despite being uniformly non-genotoxic, when administered at high dose levels to rats and mice for long periods. Three mechanisms have been proposed to explain the induction of tumours. One is based on increased production of active oxygen species due to imbalanced production of peroxisomal enzymes; it has been proposed that these reactive oxygen species cause indirect DNA damage with subsequent tumour formation. In rodents, an alternative mechanism is the promotion of endogenous lesions by sustained DNA synthesis and hyperplasia. Thirdly, it is conceivable that sustained growth stimulation may be sufficient for tumour formation. Marked species differences are apparent in response to peroxisome proliferations. Rats and mice are extremely sensitive, and hamsters show an intermediate response while guinea pigs, monkeys and humans appear to be relatively insensitive or non-responsive at dose levels that produce a marked response in rodents. These species differences may be reproduced in vitro using primary culture hepatocytes isolated from a variety of species including humans. The available experimental evidence suggests a strong association and a probable casual link between peroxisome-proliferator-elicited liver growth and the subsequent development of liver tumours in rats and mice. Since humans are insensitive or unresponsive, at therapeutic dose levels, to peroxisome-proliferator-induced hepatic effects, it is reasonable to conclude that the encountered levels of exposure to these non-genotoxic agents do not present a hepatocarcinogenic hazard to humans.(ABSTRACT TRUNCATED AT 400 WORDS)
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de Camargo JL, Tsuda H, Asamoto M, Tagawa Y, Wada S, Nagase S, Ito N. Modifying effects of chemicals on the development of liver preneoplastic placental glutathione S-transferase positive foci in analbuminemic and Sprague-Dawley rats. Toxicol Pathol 1993; 21:409-16. [PMID: 8290874 DOI: 10.1177/019262339302100410] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Nagase analbuminemic rats (NARs) were compared to the Sprague-Dawley (SD) stock in a medium-term assay system for hepatocarcinogenesis regarding their susceptibilities to the influence of chemicals on the development of glutathione S-transferase, placental form, positive (GST-P+) foci. Two weeks after initiation with diethylnitrosamine (DEN), the animals were exposed alternatively to 0.06% 3'-methyl-4-dimethyl-aminoazobenzene (3'-Me-DAB), 50 ppm DEN, 0.25% ethionine, 1% clofibrate, and 1% butylated hydroxyanisole (BHA) for a 6-wk period. Adequate controls included groups only initiated with DEN or treated with each test compound alone. For evaluation of the modifying potential of the chemicals, indices were generated by using the mean values obtained for number and area of GST-P+ foci after each treatment. Comparison between these indices suggests that SD rats were relatively more sensitive than NARs to the modifying effects of complete carcinogens (3'-Me-DAB and DEN). The strains were similarly-susceptible to the promoting influence of ethionine, a nongenotoxic carcinogen. The inhibitory influence of BHA was more intense in NARs, whereas in both strains clofibrate was associated to similarly reduced values for number and area of GST-P+ foci. The degree of susceptibility of each strain to the modifying influence of chemicals on foci development depended on the chemical agent investigated.
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Affiliation(s)
- J L de Camargo
- First Department of Pathology, Nagoya City University Medical School, Japan
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Nakanishi Y, Okamoto K, Isohashi F. Effects of chronic administration of the peroxisome proliferator, clofibrate, on cytosolic acetyl-CoA hydrolase in rat liver. Biochem Pharmacol 1993; 45:1403-7. [PMID: 8097090 DOI: 10.1016/0006-2952(93)90038-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The hypolipidemic drug ethyl chlorophenoxyisobutyrate (clofibrate) is known to induce peroxisome proliferation and to be carcinogenic after long term administration to rats and mice. We examined the effects of treatment with this drug for periods of up to 18 months on cytosolic ATP-stimulated and ADP-inhibited acetyl-CoA hydrolase in rat liver. In male Donryu albino rats on a diet containing 0.5% clofibrate, the enzyme activity increased to about 2- and 3-fold the initial level per milligram liver protein and cytosolic protein, respectively, and 2-fold per milligram DNA in 3 days, and then remained at this level for up to 18 months. The increased activity in rats receiving clofibrate for 3 months returned to control level within a week when clofibrate was withdrawn. The change in enzyme activity paralleled the change in the amount of enzyme protein determined by immunoblotting with antibody against purified acetyl-CoA hydrolase from rat liver cytosol. No liver tumors were detected macroscopically after administration of clofibrate for 18 months. However, our results suggest that cytosolic acetyl-CoA hydrolase could be an extraperoxisomal marker enzyme induced by this type of drug.
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Affiliation(s)
- Y Nakanishi
- Department of Home Economics, Kyoto University of Education, Japan
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Parzefall W, Schuppler J, Barthel G, Meyer-Rogge B, Schulte-Hermann R. Toxicological studies on a benzofurane derivative. I. A comparative study with phenobarbital on rat liver. Toxicol Appl Pharmacol 1990; 106:482-99. [PMID: 1701930 DOI: 10.1016/0041-008x(90)90343-s] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The benzofurane derivative benzbromarone (BBR) previously has led to liver tumor formation after long-term treatment of rats, but no indications of genotoxicity were detected. The present studies were designed to elucidate the mechanism(s) possibly involved in liver tumor formation by BBR. Female Wistar rats were used. Phenobarbital (PB) served as a positive control. (1) Short-term treatment (7 days) with daily doses of 2 to 100 mg/kg BBR led to adaptive responses in the liver, i.e., growth (increases in DNA, RNA, and protein) and induction of monooxygenases. These changes were also observed after feeding BBR for 8, 33, 77, and 102 weeks at doses of 2, 10, and 50 mg/kg/day but tended to weaken with time. Similar effects were obtained with PB fed at 2, 10, or 50 mg/kg/day. However, unlike PB, BBR did not enhance the expression of cytochrome P450-PB as demonstrated by immunostaining of histological liver sections. (2) BBR feeding for 102 weeks, but not for 77 weeks, produced some neoplastic liver nodules and at 50 mg/kg produced one hepatocellular carcinoma (HCC). Thus, BBR was tumorigenic in the present study, but was clearly weaker than PB which had induced liver nodules and HCCs at 77 weeks and even more markedly at 102 weeks. (3) To check for tumor-initiating activity 100 mg/kg BBR was given 14 hr after a two-thirds hepatectomy followed by promotion with PB (50 mg/kg) for 15 weeks. No phenotypically altered liver foci were detected. (4) To test for tumor-promoting activity rats received a single dose of N-nitrosomorpholine (250 mg/kg), and subsequently BBR or PB at doses of 2, 10, and 50 mg/kg/day. While PB markedly enhanced the development of neoplastic nodules and HCCs, BBR had only a weak enhancing effect on the induction of HCC, which was not dose related. gamma-glutamyl transpeptidase-positive foci dramatically increased in PB-treated animals, in contrast they showed no response after 2 and 10 mg/kg BBR and even decreased after 50 mg/kg BBR. (5) With PB changes in liver growth, monooxygenase activity, foci expansion, and tumor promotion all correlating with tumorigenesis in a quantitative manner, apparent no-observed-effect-levels are somewhat below 2 mg/kg (or 10 mg/kg for liver enlargement). (6) These studies suggest that BBR belongs to a group of nongenotoxic, growth-stimulating drugs with tumorigenic potential in rat liver. Its effects on the liver are different from those of PB, but seemed to resemble those of peroxisome proliferators, a hypothesis studied in the subsequent papers.
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Affiliation(s)
- W Parzefall
- Institut für Tumorbiologie-Krebsforschung, Universität Wien, Austria
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