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1.
Toxicol Pathol ; 40(7): 971-94, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22723046

ABSTRACT

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.


Subject(s)
Adaptation, Physiological/drug effects , Chemical and Drug Induced Liver Injury/etiology , Hepatomegaly/chemically induced , Liver/drug effects , Xenobiotics/toxicity , Adaptation, Physiological/physiology , Animals , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Congresses as Topic , Hepatomegaly/metabolism , Hepatomegaly/pathology , Humans , Liver/metabolism , Liver/pathology , Liver Diseases/metabolism , Liver Diseases/pathology , Liver Function Tests , Mice , Organ Size/drug effects , Rats , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism
2.
Carcinogenesis ; 19(9): 1521-7, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9771920

ABSTRACT

The coordinate regulation of DNA synthesis and suppression of apoptosis was investigated in a rat hepatocyte cell culture system which supports high level induction of DNA synthesis by the peroxisome proliferator, methylclofenapate (MCP) (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-931). The peroxisome proliferators are hepatocyte mitogens in chemically defined media: glucocorticoid-induced PPARalpha is linked to peroxisome proliferator mitogenesis (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-932). Phenobarbital (PB) induced moderate induction of DNA synthesis (200-300% of control), but the peak of induction was 40 h after treatment. In hepatocytes that had undergone DNA synthesis, PB increased the proportion of binucleates by 200-300%. Both PB and MCP were able to suppress apoptosis in a dose-dependent manner, while the endogenous mitogen epidermal growth factor failed to suppress apoptosis. The suppression of apoptosis by MCP was reversible; withdrawal of MCP led to rapid induction of apoptosis. The presence of hydrocortisone is required for suppression of apoptosis by peroxisome proliferators, but not for PB. MCP failed to suppress apoptosis in primary cultures of guinea-pig hepatocytes. Comparison of the stability of hepatocytes labelled with bromodeoxyuridine (BrdUrd) and [3H]thymidine revealed that approximately 40% of cells labelled with BrdUrd were lost over a period of 14 days, whereas cells labelled with thymidine remained stable over this period. Hepatocytes were therefore treated with MCP, labelled with [3H]thymidine, maintained for 14 days, and peroxisome proliferator withdrawn. While the apoptotic index in unlabelled cells was 1.7%, no apoptosis was detected in labelled cells. In order to compare the mechanism of suppression of apoptosis, hepatocytes were cultured in the presence of either PB or MCP for 14 days. When MCP was substituted for PB in cells cultured in the presence of PB, the monolayer was maintained, but when PB was used to replace MCP in cells cultured in the presence of MCP, the monolayer of hepatocytes degenerated rapidly. The results demonstrate mechanistic differences in the coordinate regulation of cell growth and apoptosis in hepatocytes by PB and MCP.


Subject(s)
Apoptosis/drug effects , Clofenapate/pharmacology , DNA/biosynthesis , Liver/drug effects , Microbodies/drug effects , Phenobarbital/pharmacology , Animals , Cell Division/drug effects , Cells, Cultured , Guinea Pigs , Liver/cytology , Male , Rats , Rats, Wistar
3.
Carcinogenesis ; 19(5): 925-31, 1998 May.
Article in English | MEDLINE | ID: mdl-9635884

ABSTRACT

Peroxisome proliferator-induced mitogenesis is believed to play a role in hepatocarcinogenesis, but it has not been possible to demonstrate high level induction of DNA synthesis by peroxisome proliferators in cultured hepatocytes. We now show that four structurally dissimilar peroxisome proliferators (methylclofenapate, Wy-14 643, tetradecyl-3-thia acetic acid and clofibrate) cause high level induction of DNA synthesis in primary cultures of rat hepatocytes, routinely 7-9 fold above control, with up to 29% of cells undergoing S-phase. Peroxisome proliferators induce DNA synthesis rapidly, with maximal response 24 h after dosing [compared with 48 h for epidermal growth factor (EGF)]; indeed, peroxisome proliferators were mitogenic in a chemically defined medium, i.e. with no added exogenous growth factors. EGF-treated hepatocytes that had undergone DNA synthesis comprised 23% binucleated cells, whereas hepatocytes induced into S-phase by peroxisome proliferators contained only 3% binucleated cells, demonstrating a distinct response of hepatocytes to peroxisome proliferators and EGF. The presence of a glucocorticoid was essential for peroxisome proliferator-induced DNA synthesis, but not for EGF-induced DNA synthesis, demonstrating that the requirement for glucocorticoids is selective for peroxisome proliferators. Hydrocortisone was shown to induce the expression of peroxisome proliferator activated receptor-alpha (PPAR alpha), and we propose that it is the glucocorticoid-induced expression of PPAR alpha that is essential for peroxisome proliferator mitogenesis. This in vitro system provides a powerful tool for investigating the mechanism and role of peroxisome proliferator-induced mitogenesis in liver growth and carcinogenesis.


Subject(s)
Glucocorticoids/pharmacology , Liver/drug effects , Microbodies/drug effects , Mitogens/pharmacology , Receptors, Cytoplasmic and Nuclear/biosynthesis , Transcription Factors/biosynthesis , Animals , Clofenapate/pharmacology , Clofibrate/pharmacology , DNA Replication/drug effects , Growth Substances/pharmacology , Male , Pyrimidines/pharmacology , Rats , Sulfides/pharmacology
4.
J Biochem Mol Toxicol ; 12(2): 79-82, 1998.
Article in English | MEDLINE | ID: mdl-9443064

ABSTRACT

The nongenotoxic carcinogens phenobarbitone (PB) and methyl clofenapate (MCP) and the hepatomitogen pregnenolone 16 alpha carbonitrile (PCN) are direct inducers of hepatic S-phase in rats, whereas the S-phase seen after partial hepatectomy is regenerative. We have investigated S-phase and immediate-early gene expression (c-myc and c-jun) in rat liver following these treatments to study the differences in gene expression associated with direct vs. regenerative responses. Both partial hepatectomy (one- and two-thirds) and mitogen treatment caused an increase in hepatic S-phase that peaked around 36 hours. Two-thirds partial hepatectomy caused the greatest increase in S-phase followed by one-third partial hepatectomy, then the mitogens PCN, MCP, and PB in that order. This order of response was also seen with c-jun and to a lesser degree with c-myc expression, suggesting that immediate-early gene expression might be linked not only to regenerative S-phase but also to direct mitogen-induced responses.


Subject(s)
Genes, Immediate-Early , Liver Regeneration , Liver/growth & development , Mitogens/pharmacology , Animals , Gene Expression/drug effects , Genes, jun , Genes, myc , Liver/drug effects , Male , Rats , Rats, Inbred F344 , S Phase
7.
Drug Metab Dispos ; 24(5): 547-54, 1996 May.
Article in English | MEDLINE | ID: mdl-8723735

ABSTRACT

Rats at day 15.5 of gestation were dosed intraperitoneally with 300 mg.kg-1 of clofibrate for three consecutive days at 24-hr intervals and were culled 24 hr after the final injection. This regime produced maximal induction of the cytochrome P4504A (CYP4A) mRNAs in the maternal liver and kidney and in 18.5-day fetal tissues. The maternal hepatic and renal CYP4A mRNA levels had risen 12- and 2-fold, respectively, above the constitutive levels seen in untreated pregnant rats at an equivalent stage of gestation. Clofibrate was capable of traversing the placenta and modulating the fetal CYP4A mRNA expression as demonstrated by a 3-fold elevation in the mRNA levels in those fetuses explanted from drug-induced mothers, compared with those fetuses removed from untreated mothers. The CYP4A mRNAs were demonstrated in the fetal liver via dot-blot and Northern blot analyses. In addition, low levels of CYP4A mRNA expression were detected in the induced placenta via Northern blot analysis. Western blot analysis revealed that the CYP4A protein levels increased in the maternal liver and in the kidney and fetal livers after exposure to clofibrate. Peroxisome proliferation, a phenomenon associated with induction of CYP4A1 expression in rodents, was demonstrated in both maternal and fetal livers, with the use of light and electron microscopy.


Subject(s)
Clofibrate/pharmacology , Cytochrome P-450 Enzyme System/genetics , Fetus/drug effects , Maternal-Fetal Exchange , Mixed Function Oxygenases/genetics , Placenta/enzymology , Animals , Base Sequence , Clofibrate/administration & dosage , Clofibrate/pharmacokinetics , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme System/biosynthesis , DNA Probes , Enzyme Induction , Female , Fetus/enzymology , Gestational Age , Hypolipidemic Agents/administration & dosage , Hypolipidemic Agents/pharmacokinetics , Hypolipidemic Agents/pharmacology , Kidney/enzymology , Liver/embryology , Liver/enzymology , Microbodies/drug effects , Mixed Function Oxygenases/biosynthesis , Molecular Sequence Data , Pregnancy , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Wistar
8.
Biochem Pharmacol ; 50(12): 2021-32, 1995 Dec 22.
Article in English | MEDLINE | ID: mdl-8849329

ABSTRACT

Lactating mothers of 7.5-day neonatal rats were injected intraperitoneally with 500 mg kg-1 clofibrate for 3 consecutive days at 24-hour intervals; 24 hours after the final injection, the maternal cytochrome P450 4A (CYP4A) mRNA levels had risen 14- and 2.5-fold above the constitutive levels of expression seen in the liver and kidney, respectively. Lactational transfer of clofibrate to the suckling 10.5-day litter was demonstrated by the 15- and 5-fold elevation observed in the neonatal hepatic and renal CYP4A mRNAs, respectively, following suckling from drug-induced mothers. A significant decrease in the relative liver weights of these neonatal pups was seen following clofibrate exposure via maternal milk, in total contrast to the normally observed increase in liver/body weight ratios of rats treated with clofibrate. Western blot analysis using a polyclonal goat anti-rat CYP4A1 antibody also demonstrated a rise in the CYP4A protein levels in both the mothers and their litters following maternal clofibrate treatment.


Subject(s)
Clofibrate/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Hypolipidemic Agents/pharmacology , Lactation , Liver/enzymology , Mixed Function Oxygenases/biosynthesis , RNA, Messenger/biosynthesis , Animals , Animals, Newborn , Animals, Suckling , Base Sequence , Blotting, Northern , Blotting, Western , Body Weight/drug effects , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme System/genetics , Enzyme Induction , Female , Kidney/drug effects , Kidney/enzymology , Liver/drug effects , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Organ Size/drug effects , Rats , Rats, Wistar
9.
Mutagenesis ; 10(6): 517-22, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8596471

ABSTRACT

A series of bacterial mutation, mammalian cell (L5178Y) gene mutation and in vitro cytogenetic assays were performed to compare the efficacy of using S9 fractions prepared from rats induced with a combination of phenobarbital (PB) and beta-naphthoflavone (beta NF), with S9 fractions from rats treated with the general enzyme inducer Aroclor 1254. Although some quantitative differences in the magnitudes of the mutagenic/clastogenic effects were observed between the two induction regimes, no qualitative differences were observed. The use of a combined PB/beta NF induction regime using oral dosing is therefore considered to be a suitable substitute for Aroclor 1254.


Subject(s)
Aroclors/toxicity , Benzoflavones/toxicity , Liver Extracts/metabolism , Mutagenicity Tests/methods , Phenobarbital/toxicity , Administration, Oral , Animals , Benzo(a)pyrene/toxicity , Benzoflavones/administration & dosage , Carcinogens/toxicity , Chromosome Aberrations , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/metabolism , Dimethylnitrosamine/toxicity , Female , Humans , Lymphocytes/cytology , Lymphocytes/drug effects , Male , Phenobarbital/administration & dosage , Rats , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics , beta-Naphthoflavone
10.
Toxicology ; 95(1-3): 51-4, 1995 Jan 06.
Article in English | MEDLINE | ID: mdl-7825190

ABSTRACT

In order to monitor the effect of the procedures required to s.c. implant osmotic pumps into rats on plasma thyroid and testosterone hormone levels, male Fischer 344 rats (8-10 weeks old) were divided into six groups of 10 rats and the groups treated in the following manner: (1) controls housed 5 per cage; (2) controls housed individually; (3) animals anaesthetised for surgery and individually housed; (4) anaesthetised, sham operated and individually housed; (5) anaesthetised, s.c. implanted with osmotic pumps containing saline and individually housed; (6) anaesthetised, s.c. implanted with osmotic pumps containing 5-bromo 2-deoxyuridine (BRDU) and individually housed. Four days after performing the surgery the study was terminated and the level of hormones in the plasma determined by radio immunoassay (RIA). Tri-iodothyronine (T3) and thyroxine (T4) plasma levels (free and total) were significantly decreased with each additional step in the procedure used for the s.c. implantation of an osmotic pump containing BRDU, when compared with the individually housed controls. Similarly, testosterone plasma levels were significantly decreased by the s.c. implantation of osmotic pumps, implying a 'stress' response might occur following implantation. These observations might need to be considered by investigators when performing toxicological research which, as part of the study, uses osmotic pumps for the delivery of the nucleotide precursor required for monitoring cells in 'S' phase.


Subject(s)
Infusion Pumps, Implantable/adverse effects , Stress, Physiological/blood , Testosterone/blood , Thyroxine/blood , Triiodothyronine/blood , Animals , DNA Replication/drug effects , Male , Rats , Rats, Inbred F344 , S Phase , Stress, Physiological/etiology , Toxicity Tests
11.
Hum Exp Toxicol ; 13(11): 764-75, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7857696

ABSTRACT

The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays. It gave a negative response in each of the following assays: mutagenicity to S. typhimurium and E. coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays. The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level. These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain. These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP). Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase). Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity. These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP. The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP. In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.


Subject(s)
Carcinogens/toxicity , Clofenapate/toxicity , Microbodies/drug effects , Mutagens/toxicity , Pyrimidines/toxicity , Animals , Bone Marrow/drug effects , CHO Cells , Chromosome Aberrations/genetics , Cricetinae , Humans , In Vitro Techniques , Liver/drug effects , Lymphocytes/drug effects , Male , Mice , Mice, Transgenic , Micronucleus Tests , Rats , Species Specificity
12.
Hum Exp Toxicol ; 13 Suppl 2: S1-117, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7857698

ABSTRACT

In this review we have evaluated the relationship between peroxisome proliferation and hepatocarcinogenesis. To do so, we identified all chemicals known to produce peroxisome proliferation and selected those for which there are data (on peroxisome proliferation and hepatocarcinogenesis) which meet certain criteria chosen to facilitate comparison of these phenomena. The summarised data and definition of the methodology used has been collected in appendices. These comparisons enabled us to evaluate the relationship between these phenomena using reliable data. As there is a good correlation between them, we further explored the mechanisms of action that have been proposed (direct genotoxic activity, production of hydrogen peroxide, cell proliferation and receptor activation). The relationship between these events in other species, including humans, was also reviewed and finally an overview of the assessment of human hazard is presented in section IX. Some of the first chemicals which were shown to produce peroxisome proliferation were also hepatocarcinogens whose carcinogenicity could not be readily explained by genotoxic activity. This raised the suggestion that the unusual phenomenon of peroxisome proliferation was intricately linked to the carcinogenic activity of these agents. Three questions have exercised the attention of regulatory, industrial and academic toxicology since then; are chemicals which elicit peroxisome proliferation in the liver actually a coherent class of chemical carcinogens?; does the early biological phenomenon of peroxisome proliferation have real predictive value for and mechanistic association with rodent carcinogenesis?; and what hazard/risk do these agents pose to humans that may be exposed to them? Whether peroxisome proliferators are indeed a discrete class of rodent carcinogens would appear to be the single, most important question. If so, then the assumptions and procedures relevant to human hazard and risk assessment should be applied to the class and should be essentially generic; if not, each chemical should be considered independently. Our critical analysis of the published data for over 70 agents which have been shown to possess intrinsic ability to induce peroxisome proliferation in the livers of rodents has led to the conclusion that there exists a strong correlation between peroxisome proliferation as n early effect in the liver and hepatocarcinogenicity in chronic exposure studies. An almost perfect correlation was observed between the induction of peroxisomes in the rodent liver and the eventual appearance of tumours following chronic exposure The few exceptions to this were largely explainable (section II).(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Carcinogens/toxicity , Liver Neoplasms/chemically induced , Liver/drug effects , Microbodies/drug effects , Animals , Binding, Competitive , Biomarkers, Tumor/metabolism , Carcinogens/chemistry , Cell Division/drug effects , Cell Division/genetics , Cell Division/physiology , DNA Damage/drug effects , DNA Repair/drug effects , Databases, Factual , Female , Humans , Hydrogen Peroxide/metabolism , Liver/cytology , Male , Mice , Microbodies/enzymology , Microbodies/metabolism , Rats , Species Specificity , Structure-Activity Relationship , Transcription, Genetic/genetics
13.
Biochem Pharmacol ; 47(5): 763-73, 1994 Mar 02.
Article in English | MEDLINE | ID: mdl-8135852

ABSTRACT

The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). In monkeys there were no sex-differences in untreated, phenobarbitone (PB)- or beta-naphthoflavone (BNF)-treated animals in AROD activities, or in individual P450 proteins detected by immunoblotting. Basal MROD and EROD activities varied by less than 7-fold between the five species, but the comparative pattern of basal MROD, EROD, PROD and BROD activities (the "MEPB profile") was very species-specific, with monkeys being similar to rats but different from man, mouse and hamster. The induction of AROD activities by PB and BNF was also highly species-specific. Monkeys expressed constitutive proteins immunorelated to the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A sub-families (human CYP2A6 cross-reacted with the anti-rat CYP2B1 antibodies used, and so CYP2A and CYP2B forms could not be separately identified in the monkey). Single constitutive immunoblot bands were identified in monkey for CYP1A (54 kDa), CYP2A/CYP2B (51 kDa) and CYP3A (51 kDa), respectively, but two strong (51 and 52 kDa) plus two weak (49 and 49.5 kDa) bands were shown for CYP2C. Human liver expressed CYP1A2 (54 kDa), CYP2A6 (51 kDa), CYP3A4 (50.5 kDa) and three CYP2C9-immunorelated protein bands (48, 50 and 54 kDa). In monkeys BNF induced the 54 kDa CYP1A protein and CYP1A-dependent MROD, EROD and PROD activities (18-, 15- and 6-fold increases in activity, respectively), whereas PB strongly induced the 51 kDa CYP2A/CYP2B protein but did not induce PROD activity. PB also induced non-constitutive CYP2A/CYP2B protein bands at 49 and 52 kDa in some monkeys. BROD activity was induced less that four-fold by either PB or BNF in monkeys. In conclusion, cynomolgus monkeys expressed a range of constitutive CYP1A, CYP2A or CYP2B, CYP2C and CYP3A proteins similar to man, and a range of AROD monooxygenase reaction rates similar to both man and rat, but the basal MEPB profile of AROD activities in monkeys was more similar to rat than to man. MROD and EROD were good measures of CYP1A induction by polycyclic aromatic hydrocarbons in cynomolgus monkeys, but neither PROD nor BROD were indices of CYP2B induction by PB.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/enzymology , Adult , Animals , Cricetinae , Cytochrome P-450 CYP1A1 , Cytochrome P-450 CYP2B1 , Dealkylation , Enzyme Induction , Female , Humans , Immunoblotting , Macaca fascicularis , Male , Mesocricetus , Mice , Mice, Inbred C57BL , Middle Aged , Oxidoreductases/metabolism , Rats , Rats, Sprague-Dawley , Species Specificity
14.
Biochem Pharmacol ; 45(10): 2045-53, 1993 May 25.
Article in English | MEDLINE | ID: mdl-7685601

ABSTRACT

We have analysed the induction of microsomal and peroxisomal proteins and their RNAs after treatment of hepatocytes with the peroxisome proliferator, clofibric acid, in vitro and in vivo. After treatment of hepatocytes with 1 mM clofibric acid for 4 days, P450 4A1 RNA is induced 500-fold, and acyl-CoA oxidase and P450 2B1 280-fold, relative to control cultures. These RNAs are detectably induced after administration of 25 microM clofibric acid, and show a similar induction response with increasing doses of clofibric acid. Western blot analysis of the P450 4A and bifunctional enzyme (BFE) proteins showed that both were induced in parallel with increasing doses of clofibric acid, over a range of 25 microM-1 mM. The distribution of the induced proteins was examined by immunocytochemistry. Increasing doses of clofibric acid led to an increase in the average intensity of staining for both proteins throughout the hepatocyte population. There was, however, a graded variation between hepatocytes in the intensity of staining, both for P450 4A and BFE proteins. The heterogeneity in response of the hepatocyte population in vitro may be related to differential sensitivity of hepatocytes to induction in vivo. Therefore, rats were dosed with 0, 50 or 300 mg/kg of clofibric acid for 4 days by gavage, and the livers were examined by immunocytochemistry. After 50 mg/kg of clofibric acid, both P450 4A and BFE were induced mainly in zones 3 and 2 of the liver acinus. However, after 300 mg/kg of clofibric acid, staining for both proteins was strong and homogenous throughout the liver acinus. Thus, hepatocytes from zones 3 and 2 of the acinus are differentially responsive to induction by clofibric acid.


Subject(s)
Clofibric Acid/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Microbodies/enzymology , 3-Hydroxyacyl CoA Dehydrogenases/biosynthesis , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Acyl-CoA Oxidase , Animals , Base Sequence , Blotting, Western , Cytochrome P-450 CYP2B1 , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme System/genetics , Enoyl-CoA Hydratase/biosynthesis , Enoyl-CoA Hydratase/genetics , Enzyme Induction/drug effects , Immunohistochemistry , Isomerases/biosynthesis , Isomerases/genetics , Liver/cytology , Liver/drug effects , Liver/enzymology , Male , Microbodies/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Oxidoreductases/biosynthesis , Oxidoreductases/genetics , Peroxisomal Bifunctional Enzyme , RNA/biosynthesis , RNA/genetics , Rats , Rats, Wistar
15.
Biochem Pharmacol ; 45(4): 827-32, 1993 Feb 24.
Article in English | MEDLINE | ID: mdl-8452557

ABSTRACT

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.


Subject(s)
Carnitine Acyltransferases/antagonists & inhibitors , Diethylhexyl Phthalate/pharmacology , Mitochondria, Liver/drug effects , Phthalic Acids/pharmacology , Animals , Binding Sites , Binding, Competitive , Caprylates/metabolism , Diethylhexyl Phthalate/metabolism , Fatty Acids/metabolism , Glucosides , Guinea Pigs , Humans , Kinetics , Male , Mice , Microbodies/drug effects , Mitochondria, Liver/enzymology , Palmitates/metabolism , Rats
16.
Toxicology ; 77(1-2): 81-90, 1993 Jan 29.
Article in English | MEDLINE | ID: mdl-8442021

ABSTRACT

Male rats and mice were administered chlorinated paraffins (CPs) by daily gavage in corn oil for 14 days. Chlorowax 500C (short chain CP with 58% chlorination), Cereclor 56L (short chain CP with 56% chlorination) and Chlorparaffin 40G (medium chain CP with 40% chlorination) were the CPs studied at dose levels of 0, 10, 50, 100, 250, 500 and 1000 mg/kg for both rats and mice. The no effect levels for hepatic peroxisome proliferation for the above chemicals, as determined by the CN- insensitive palmitoyl co-enzyme A beta-oxidation (PCO) assay, were calculated as 184, 600 and 473 mg/kg and 180, 120 and 252 mg/kg for rats and mice, respectively, whilst those for percent liver weight/body weight were calculated as 74, 51 and 31 mg/kg and 215, 70 and 426 mg/kg for rats and mice, respectively. The short chain CPs were more potent peroxisome proliferators than the medium chain CP, with the mouse proving to be more responsive than the rat. Rats administered the highest dose of CPs showed a depressed plasma thyroxine (T4) level, with a concomitant increase in the plasma concentrations of thyroid stimulating hormone (TSH). The decreased plasma T4 levels appeared to be the result of increased T4 glucuronidation.


Subject(s)
Hydrocarbons, Chlorinated/toxicity , Liver/enzymology , Thyrotropin/blood , Thyroxine/blood , Animals , Body Weight/drug effects , Dose-Response Relationship, Drug , Hydrocarbons, Chlorinated/administration & dosage , Male , Mice , Mice, Inbred Strains , Microbodies/drug effects , Organ Size/drug effects , Paraffin/analogs & derivatives , Paraffin/toxicity , Rats , Rats, Sprague-Dawley
17.
Eur J Biochem ; 206(3): 979-86, 1992 Jun 15.
Article in English | MEDLINE | ID: mdl-1376690

ABSTRACT

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Mixed Function Oxygenases/biosynthesis , Organ Specificity , Oxidoreductases/biosynthesis , Acyl-CoA Oxidase , Animals , Clofenapate/pharmacology , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme System/genetics , Enzyme Induction , Kidney/enzymology , Liver/enzymology , Lung/enzymology , Male , Mixed Function Oxygenases/genetics , Muscles/enzymology , Oxidoreductases/genetics , RNA/analysis , RNA/biosynthesis , Rats , Testis/enzymology
18.
Biochem Pharmacol ; 43(10): 2129-34, 1992 May 28.
Article in English | MEDLINE | ID: mdl-1599500

ABSTRACT

Identification of the proximate peroxisome proliferator(s) derived from di (2-ethylhexyl) adipate (DEHA) has been achieved using primary hepatocyte cultures derived from different species and cyanide-insensitive fatty acyl CoA oxidase (PCO) as a marker enzyme for peroxisome proliferation. In rat and mouse hepatocytes, the parent compound (DEHA) had no effect on peroxisomal beta-oxidation, but primary metabolites of DEHA, mono (2-ethylhexyl) adipate (MEHA) and 2-ethylhexanol (EH), were approximately equipotent in PCO induction (5-fold at 0.5 mM final concentration). The secondary metabolite of DEHA, 2-ethylhexanoic acid (EHA), was in both species the most potent peroxisome proliferator (25- and 9-fold induction in mice and rats, respectively, at 1 mM final concentration). At 2 mM final concentration a tertiary metabolite of DEHA, 2-ethyl-5-hydroxyhexan-1-oic acid, was less effective in mouse and rat hepatocytes at inducing PCO (15- and 5-fold, respectively). 2-Ethyl-5-oxohexan-1-oic acid and 2-ethylhexan-1,6-dioic acid had little effect (2-3-fold in both rat and mouse hepatocytes). Thus, EHA was identified as the proximate peroxisome proliferator of DEHA and mouse hepatocytes were approximately twice as sensitive as rat hepatocytes to peroxisome proliferation due to MEHA, EH and EHA. We investigated further species differences in response to peroxisome proliferators by using guinea pig and marmoset primary hepatocyte culture. None of the chemicals studied stimulated peroxisomal beta-oxidation in these species up to a final concentration of 2 mM. Higher concentrations lead to cytotoxicity. This lack of sensitivity of guinea pig and marmoset hepatocytes is in agreement with previous studies with di (2-ethylhexyl) phthalate metabolites, suggesting the absence of a threat of hepatocarcinogenic damage to these species and confirming that primary hepatocytes cultures are useful models for investigating the phenomenon of peroxisome proliferation.


Subject(s)
Adipates/pharmacology , Liver/drug effects , Microbodies/drug effects , Plasticizers/pharmacology , Adipates/metabolism , Animals , Callithrix , Cells, Cultured , Guinea Pigs , Liver/ultrastructure , Male , Mice , Models, Chemical , Oxidation-Reduction , Phthalic Acids/metabolism , Rats , Species Specificity
19.
Mutagenesis ; 7(3): 175-7, 1992 May.
Article in English | MEDLINE | ID: mdl-1602970

ABSTRACT

A working party was set up by the UK Environmental Mutagen Society to consider alternatives to Aroclor 1254 (Aroclor)-induced S9 in in vitro genotoxicity assays, with the aims of considering whether a replacement for Aroclor in its role in general screening assays could be readily identified. The working party concluded that there was sufficient support in the literature to justify the use of an appropriate phenobarbital/beta-naphthoflavone regime as an acceptable alternative to Aroclor.


Subject(s)
Aroclors , Mutagenicity Tests , Benzoflavones , Liver Extracts , Mutagenicity Tests/methods , Phenobarbital , United Kingdom , beta-Naphthoflavone
20.
Biochem J ; 282 ( Pt 3): 635-8, 1992 Mar 15.
Article in English | MEDLINE | ID: mdl-1313227

ABSTRACT

The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting a regional pattern in the intact liver and a varied response to phenobarbital in isolated cultured hepatocytes. We report that P450 2B1/2 immunostaining of hepatocytes isolated from the perivenous liver region and cultured in the presence of phenobarbital is much stronger than that of cells identically treated but isolated from the periportal region. P450 2B1 mRNA, quantified by a sensitive and specific RNAase protection assay, is also preferentially induced in perivenous hepatocytes, demonstrating that the difference in induced expression is at the pretranslational level. Our results suggest that perivenous and periportal hepatocytes are differentially imprinted to retain regiospecific factors governing their inducibility after isolation.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Isoenzymes/biosynthesis , Liver/enzymology , Animals , Base Sequence , Blotting, Western , Cells, Cultured , Cytochrome P-450 Enzyme System/genetics , Enzyme Induction , Immunohistochemistry , Isoenzymes/genetics , Liver/blood supply , Liver/cytology , Male , Microbial Collagenase/pharmacology , Molecular Sequence Data , Phenobarbital/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Veins
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