RESUMO
The hepatocarcinogenicity of 2,4-dinitrotoluene [(2,4-DNT) CAS: 121-14-2], 2,6-DNT (CAS: 606-20-2), and a representative technical-grade DNT (TDNT) containing 76% 2,4-DNT and 18% 2,6-DNT was studied in male F344 rats. Rats were fed diets containing 2,4-DNT, 2,6-DNT, or TDNT at concentrations that resulted in doses of 27 mg/kg/day for 2,4-DNT, 7 or 14 mg/kg/day for 2,6-DNT, and 35 mg/kg/day for TDNT. The carcinogenic effects were evaluated after 1 year of treatment. Administration of 2,6-DNT produced hepatocellular carcinomas in 100 and 85% of animals receiving 14 and 7 mg/kg, respectively. In contrast to the 2,6-DNT results, feeding of 2,4-DNT for 1 year caused no hepatic tumors. Treatment with both isomers (TDNT) resulted in a 47% incidence of hepatocellular tumors. The majority of tumors had a trabecular pattern, and pulmonary metastases were present in the 14- and 7-mg/kg 2,6-DNT-fed groups. These results have demonstrated that 2,6-DNT is a potent and complete hepatocarcinogen and that 2,4-DNT, under these conditions, is nonhepatocarcinogenic. In addition, these data indicate that 2,6-DNT accounts for the majority of the carcinogenic activity of TDNT.
Assuntos
Carcinógenos , Dinitrobenzenos/toxicidade , Neoplasias Hepáticas Experimentais/patologia , Nitrobenzenos/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Epóxido Hidrolases/metabolismo , Isomerismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Microssomos Hepáticos/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Fenobarbital/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.
Assuntos
Reparo do DNA/efeitos dos fármacos , Dimetilnitrosamina/farmacologia , Ditiocarb/farmacologia , Fígado/efeitos dos fármacos , Nitrosaminas/farmacologia , Tiocarbamatos/farmacologia , Aminoacetonitrila/farmacologia , Animais , Citocromos/metabolismo , DNA , Dimetilnitrosamina/metabolismo , Técnicas In Vitro , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Metilação , Microssomos Hepáticos/metabolismo , Necrose , Ratos , Fatores de TempoRESUMO
In order to characterize the promoting activity of the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,463), male F344 rats which received a single 150-mg/kg dose of diethylnitrosamine (DEN) were fed 0.1% WY-14,643 or 0.05% phenobarbital in the diet for 11, 22, or 54 wk. WY-14,643 promoted the development of ATPase-deficient foci but not GGTase-positive or G6Pase-deficient foci, in contrast to phenobarbital which promoted development of foci detected by all three markers. The mode of promotion of ATPase-deficient foci by WY-14,643 was distinctly different from that of phenobarbital. WY-14,643 primarily increased mean volume of foci at 11 and 22 wk, while phenobarbital primarily increased the numerical density of foci at these time points. At 54 wk, the yield of hepatic neoplasms per liver was higher in rats fed WY-14,643 than in rats fed phenobarbital. To evaluate the possibility that the promotional activity of WY-14,643 was more effective at a later stage in hepatocarcinogenesis, rats receiving a dose of DEN and then phenobarbital in the diet for 11 wk were changed to a diet containing WY-14,643 for an additional 11 or 43 wk. However, WY-14,643 feeding from wk 11 to 22 caused a reduction in volume density of ATPase-deficient foci relative to the volume density of foci at 11 wk. In addition, feeding WY-14,643 from wk 11 to 54 caused similar yields of hepatic neoplasms whether or not phenobarbital was fed for the initial 11 wk. WY-14,643 induced hepatic peroxisome proliferation as indicated by palmitoyl CoA oxidase activity regardless of prior treatment with DEN and/or phenobarbital. The yield of neoplasms in rats not receiving DEN was greater in rats fed WY-14,643 for wk 11 to 54 than in rats fed WY-14,643 for wk 1 to 54. In summary, the peroxisome proliferator WY-14,643 was a more efficient promoter of hepatocarcinogenesis in DEN-initiated rats than phenobarbital. The promotional activity of WY-14,643, when evaluated by stereological analysis and by changing promoters, is distinct from that of phenobarbital, perhaps suggesting different cellular and/or molecular mechanisms of promotion. Understanding the role of promotion by WY-14,643 and other peroxisome proliferators may be important in understanding the mechanism of their hepatocarcinogenicity.
Assuntos
Anticolesterolemiantes/toxicidade , Carcinógenos , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Fenobarbital/toxicidade , Lesões Pré-Cancerosas/induzido quimicamente , Pirimidinas/toxicidade , Adenosina Trifosfatases/metabolismo , Animais , Dietilnitrosamina/toxicidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microcorpos/efeitos dos fármacos , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Endogâmicos F344 , Fatores de Tempo , gama-Glutamiltransferase/metabolismoRESUMO
The microcirculation of nodules (0.5 to 10 mm in diameter) from diethylnitrosamine-treated rats was studied in perfused livers. Microlight guides were placed on nodules and surrounding tissue on the capsular surface of the liver to measure fluorescence due to fluorescein-dextran (12 microM), a dye confined to the vascular space, infused via the hepatic artery and portal vein separately or simultaneously. The fluorescence increase due to fluorescein-dextran infusion via the artery and vein simultaneously was used to compare vascular space in nodules with that of surrounding tissue. The vascular space of nodules less than 1 mm in diameter was only about one-half as large as that of surrounding tissue. In contrast, in nodules 1 to 2 mm in diameter, the vascular space was similar to values from surrounding tissue. This was largely due to an increase in the fluid entering via the artery. As nodules grew from 2 to 10 mm in diameter, the vascular space decreased as a function of nodule size to 40% of surrounding tissue. The sum of fluorescence increases due to fluorescein-dextran infused via the artery and vein separately always equalled values obtained from simultaneous infusions. From these measurements, the fraction of vascular fluid observed by the microlight guide that entered the liver via the artery was calculated. In tissue surrounding nodules, fluid entering from the artery was 19% of the total, a value approximating the fraction of fluid pumped into the liver via the artery (25%). The percentage of fluid in the nodule that entered the liver via the hepatic artery increased progressively to 100% of the total as nodules grew from 2 to 10 mm in diameter. Thus, nodules become increasingly dependent on the hepatic artery and less dependent on blood supply via the portal vein as they grow.
Assuntos
Circulação Hepática/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Animais , Dietilnitrosamina , Fluorescência , Fígado/patologia , Neoplasias Hepáticas Experimentais/patologia , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/patologia , Ratos , Ratos Endogâmicos F344RESUMO
The mechanism of hepatocarcinogenesis caused by peroxisome proliferators (PP) is poorly understood, making it difficult to predict the carcinogenicity of PP to rodents or other species. It has been suggested that the carcinogenic potential of individual PP in rodents is correlated with the degree of PP-induced hepatic peroxisome proliferation. To evaluate this possible correlation, di(2-ethylhexyl)phthalate (DEHP) at 1.2% and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) at 0.1% were fed to male F-344 rats for up to 365 days and hepatocytic peroxisome proliferation and DNA replication were measured. All rats fed Wy-14,643 for 365 days had numerous grossly visible nodules in comparison to none in the livers of DEHP-fed or control rats. Despite this difference in the induction of tumors, both DEHP and Wy-14,643 increased the peroxisomal volume density 4- to 6-fold from 8 to 365 days of treatment. Peroxisomal beta-oxidation enzyme activities were increased 8-fold by both DEHP and Wy-14,643 after 18 days. At later time points (77 to 365 days), these enzyme activities were about 25% higher in livers of Wy-14,643- than DEHP-fed rats. DEHP or Wy-14,643 increased absolute liver weights 50 to 75% above controls after 18 to 365 days of feeding. Labeling of hepatocyte nuclei with a single injection of tritiated thymidine revealed a rapid burst in replicative DNA synthesis in both DEHP and Wy-14,643-fed rats, with a return to control levels by 4 days. Additional rats were implanted with 7-day osmotic pumps containing tritiated thymidine. With this more extended method of labeling a 5- to 10-fold increase in replicative DNA synthesis was observed in rats receiving Wy-14,643 for 39 to 365 days as compared to DEHP-fed rats or controls. In conclusion, when performed under conditions similar to the tumorigenicity studies, the degree of peroxisome proliferation correlated poorly with the relative hepatocarcinogenicity of DEHP and Wy-14,643. However, a strong correlation was observed between the relative hepatocarcinogenicity of DEHP and Wy-14,643 and the ability to induce a persistent increase in replicative DNA synthesis. These data emphasize the possible importance of cell replication in the mechanism of PP-induced hepatocarcinogenesis.
Assuntos
DNA/biossíntese , Dietilexilftalato/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Microcorpos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Pirimidinas/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The diffusion of H2O2 into the cytoplasm from peroxisomes during high rates of peroxisomal beta oxidation of fatty acids was studied in perfused livers from rats treated with the hepatocarcinogenic peroxisome proliferator, nafenopin. Efflux of oxidized glutathione (GSSG) into the bile was used as a measure of increased H2O2 supply for cytoplasmic glutathione peroxidase. Male F-344 rats were given methylcellulose vehicle or nafenopin (80 mg/kg/day) by gavage for 5-8 days and livers perfused in situ with Krebs-Henseleit buffer containing 50 microM taurocholate and 0.75 g/100 ml albumin. In livers from fed, vehicle-treated or fed, nafenopin-treated rats basal rates of GSSG efflux were about 60 nmol/g/h. Subsequent infusion of 350 microM lauric acid, an excellent substrate for peroxisomal beta-oxidation, had no effect on GSSG efflux. To maximize fatty acid oxidation rats were fasted 16-20 h. In livers from fasted, nafenopin-treated rats the basal rate of GSSG efflux was 384 +/- 85 (SE) nmol/g/h (n = 8). Subsequent infusion of lauric acid increased the rate to 940 +/- 138 nmol/g/h. In livers from fasted, vehicle-treated rats lauric acid caused GSSG efflux to increase slightly from 104 +/- 14 to 286 +/- 37 nmol/g/h (n = 9). Efflux of reduced glutathione in bile was similar in livers from fasted, vehicle-treated (163 +/- 15 nmol/g/h) and fasted, nafenopin-treated rats (135 +/- 17 nmol/g/h) and decreased about 30% with lauric acid infusion. N-Octanoyl and oleoyl coenzyme A were excellent substrates for cyanide-insensitive NAD+ reduction in liver homogenates from fasted, nafenopin-treated rats whereas n-butyl, linoleoyl, and arachidonyl coenzyme A were poor substrates. Infusion of octanoate and oleate caused large increases in GSSG efflux from perfused livers from fasted, nafenopin-treated rats. In contrast, butyrate, linoleate, and arachidonate had no effect on GSSG efflux from livers from fasted, nafenopin-treated rats. Octanoate, oleate, linoleate, butyrate, and arachidonate had no effect on GSSG efflux from livers from fasted, vehicle-treated rats. Infusion of 2-bromooctanoate (600 microM) completely blocked lauric acid-induced increases in GSSG efflux and acetoacetate and beta-hydroxybutyrate production in livers from fasted, nafenopin-treated rats. Infusion of 1-3-bis(2-chloroethyl)-1-nitrosourea reduced glutathione reductase activity by 90% but did not alter lauric acid-induced increases in GSSG efflux or ketogenesis in livers from fasted, nafenopin-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Glutationa/metabolismo , Fígado/metabolismo , Microcorpos/efeitos dos fármacos , Nafenopina/farmacologia , Oxirredutases/fisiologia , Propionatos/farmacologia , Acil-CoA Oxidase , Animais , Caprilatos/farmacologia , Carmustina/farmacologia , Peróxido de Hidrogênio/metabolismo , Ácidos Láuricos/farmacologia , Masculino , Oxirredução , Perfusão , Ratos , Ratos Endogâmicos F344 , Especificidade por SubstratoRESUMO
The portal circulation of diethylnitrosamine-initiated nodules (0.5 to 7 mm in diameter) was studied in rat livers perfused exclusively via the portal vein. Microlight guides were placed on normal and nodular tissue on the capsular surface of the liver to measure pyridine nucleotide fluorescence (366 leads to 450 nm). When oxygen tension of the inflow perfusate was lowered, fluorescence in both normal tissue and small nodules (less than 2 mm in diameter) increased sharply due to the reduction of pyridine nucleotides, indicating previous normoxia. In contrast, similar manipulations did not increase fluorescence in nodules greater than 2 mm in diameter, demonstrating that nicotinamide adenine dinucleotide was reduced maximally previously; i.e., the nodules were anoxic. Direct measurements of nodule oxygen concentrations with a miniature oxygen electrode confirmed these results. 7-Hydroxycoumarin or fluorescein could be detected with micro-light guides in normal tissue and nodules less than 2 mm in diameter but not in nodules greater than 2 mm in diameter. Furthermore, fluorescent microscopy indicated an absence of fluorescein in nodules greater than 2 mm in diameter. Therefore, with four independent optical and polarographic techniques, we have demonstrated reduced portal circulation in nodules greater than 2 mm in diameter; however, smaller nodules could not be differentiated from normal tissue.
Assuntos
Circulação Hepática/efeitos dos fármacos , Neoplasias Hepáticas/induzido quimicamente , Consumo de Oxigênio , Sistema Porta/fisiopatologia , Lesões Pré-Cancerosas/induzido quimicamente , 2-Acetilaminofluoreno , Animais , Dietilnitrosamina , Fluoresceína , Fluoresceínas , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Oxigênio/análise , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Endogâmicos F344RESUMO
Fluorescein-isothiocyanate dextran (FITC-dextran), a dye confined to the vascular space, was infused via the hepatic artery and portal vein into perfused livers from fed rats treated with diethylnitrosamine for 4 to 5 months. Fluorescence due to FITC-dextran was detected with fiberoptic microlight guides placed on surface nodules of about 5 mm in diameter. Nodules were categorized into groups with normal and compromised microcirculation based on their fluorescence following infusion of FITC-dextran. Similar results were obtained when nodules were classified based on reflectance of trypan blue. Despite compromised microcirculation, ATP and ADP levels as well as ATP/ADP ratios were comparable in both groups of nodules; however, AMP was elevated in FITC-dextran-negative nodules (i.e., those with compromised microcirculation). Nodules with compromised microcirculation also contained higher glucose and lactate levels than nodules that were well perfused; however, glycogen was five times lower than in FITC-dextran-positive nodules. Fasting reduced ATP/ADP ratios in poorly perfused nodules in comparison to well-perfused nodules. In perfused livers from fed rats where glycogen was high, however, ATP/ADP ratios and rates of ATP depletion during ischemia were the same in well-perfused and poorly perfused nodules. Products of glycogen breakdown (e.g., glucose and lactate) were elevated in nodules from livers of fed but not fasted rats. The results indicate that alteration of perfusion of hepatic nodules does not change ATP levels nor the capacity of nodules to utilize high energy phosphate during anoxia. Thus, near normal energy status is maintained from glycogen metabolism in poorly perfused nodules via glycolysis. Since basal ATP content and utilization is comparable in well and poorly perfused nodules, compromised energy status is unlikely to explain selection of nodules that regress to near normal hepatocytes.
Assuntos
Nucleotídeos de Adenina/metabolismo , Metabolismo dos Carboidratos , Fluoresceína-5-Isotiocianato/análogos & derivados , Circulação Hepática , Neoplasias Hepáticas Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Dextranos , Dietilnitrosamina , Fluoresceínas , Corantes Fluorescentes , Glucose/metabolismo , Lactatos/metabolismo , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Masculino , Microcirculação/fisiopatologia , Lesões Pré-Cancerosas/irrigação sanguínea , Lesões Pré-Cancerosas/induzido quimicamente , Ratos , Ratos Endogâmicos F344 , Espectrometria de FluorescênciaRESUMO
A number of plasticizers and lipid-lowering drugs induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. In this study, seven structurally dissimilar peroxisome proliferating agents were shown to uncouple oxidative phosphorylation in isolated rat liver mitochondria. For example, perfluorooctanoate (0.5 mM) increased succinate-induced (state 4) mitochondrial respiration by over 50% while stimulation of state 3 respiration with ADP was minimal (i.e., uncoupling occurred). Interestingly, compounds which are potent carcinogens in vivo (e.g., Wy-14,643 and perfluorooctanoate) were more powerful uncouplers of oxidative phosphorylation in vitro than weak tumor-causing agents (e.g., valproate). Uncoupling also occurred in vivo. Basal rates of oxygen uptake in perfused livers from chronically treated rats were increased from 137 +/- 7 mumol g-1/h in pair-fed controls to 153 +/- 5 mumol g-1/h after 2.5 months of feeding Wy-14,643 (0.1% w/v in diet). Concomitantly, rates of urea synthesis from ammonia, a process highly dependent on ATP supply, were reduced almost completely from 104 +/- 10 mumol g-1/h to 13 +/- 6 mumol g-1/h. Bile flow, another energy-dependent process, was also reduced significantly by treatment with Wy-14,643 in vivo for 24 h. Taken together, these data indicate that energy supply for cellular processes such as urea synthesis and bile flow was disrupted in vivo due to uncoupling of oxidative phosphorylation by Wy-14,643. It is proposed that peroxisomal proliferators accumulate in the liver where they uncouple mitochondrial oxidative phosphorylation and interfere with cellular energetics.
Assuntos
Carcinógenos/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Pirimidinas/farmacologia , Animais , Bile/metabolismo , Masculino , Microcorpos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Modelos Biológicos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Ureia/metabolismoRESUMO
Female F-344 rats were fed a diet containing up to 1.2% di(2-ethylhexyl)phthalate (DEHP) for 2 years, which previously resulted in hepatocarcinogenesis under bioassay conditions. Peroxisome proliferation, decreased glutathione peroxidase activity, and lipofuscin accumulation were all associated with prolonged feeding of 1.2% DEHP and induction of hepatic neoplasia. These results establish a potential role for persistent peroxisome proliferation and oxidative injury in the hepatocarcinogenicity of dietary DEHP. Increased hepatocellular proliferation and hepatomegaly were not detected. DEHP feeding did not increase the volume density of basophilic or ATPase-deficient foci of altered hepatocytes, suggesting that these lesions are not suitable indicators of DEHP carcinogenesis.
Assuntos
Dietilexilftalato/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Fígado/efeitos dos fármacos , Microcorpos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Feminino , Lipofuscina/análise , Oxirredução , Ratos , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Elevations of oxidatively modified DNA bases have been associated with a variety of carcinogens and tumor promoters, and implicated in causation of cancer. Since carcinogen exposure can induce cell proliferation, the relationship between induction of cell proliferation and levels of DNA base oxidation was examined. Cell proliferation was induced in livers of male F344 rats by stimuli of either regeneration or hyperplasia. Levels of DNA base oxidation were evaluated by measuring 8-OH-deoxyguanosine/deoxyguanosine (8-OHdG/dG) ratios by HPLC in enzymatic digests of DNA isolates. Despite induction of cell proliferation, hepatic levels of 8-OHdG/dG were not increased at 1, 2, 3 or 5 days after any of these treatments. Results of the present work suggest that the mechanism of elevated levels of DNA base oxidation is not directly related to induction of cell proliferation.
Assuntos
Desoxiguanosina/análogos & derivados , Regeneração Hepática/fisiologia , Fígado/patologia , 8-Hidroxi-2'-Desoxiguanosina , Animais , Tetracloreto de Carbono/administração & dosagem , Divisão Celular , Desoxiguanosina/metabolismo , Hiperplasia/induzido quimicamente , Hiperplasia/metabolismo , Fígado/metabolismo , Masculino , Oxirredução , Fenobarbital , Pirimidinas , Ratos , Ratos Endogâmicos F344RESUMO
The potent peroxisome proliferator hepatocarcinogens WY-14,643, BR-931, and nafenopin, as well as mono(ethylhexyl)phthalate (MEHP), the principle metabolite of the weaker hepatocarcinogen di(2-ethylhexyl)phthalate) (DEHP), were evaluated in the in vitro rat hepatocyte DNA repair assay by quantitative autoradiography. None of these carcinogens induced unscheduled DNA synthesis (UDS). These results failed to confirm the previously reported induction of UDS by WY-14,643 and BR-931 as determined by nuclear liquid scintillation counting. Hydroxyurea (HU, 10 mM) is commonly employed to inhibit replicative DNA synthesis (RDS) when using scintillation counting techniques for UDS. Autoradiographs revealed incomplete inhibition of RDS by HU.
Assuntos
Carcinógenos , Reparo do DNA/efeitos dos fármacos , Fígado/efeitos dos fármacos , Microcorpos/efeitos dos fármacos , Animais , Autorradiografia , Divisão Celular/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Dietilexilftalato/toxicidade , Técnicas In Vitro , Fígado/metabolismo , Masculino , Nafenopina/toxicidade , Pirimidinas/toxicidade , Ratos , Ratos Endogâmicos F344RESUMO
Cell proliferation (S phase response) in archival liver tissues of partially hepatectomized rats was determined via proliferating cell nuclear antigen (PCNA) immunohistochemistry. These results were compared with the S phase response assessed previously in the same tissues via tritiated thymidine (Tdr) autoradiography. The effect of prolonged tissue fixation on PCNA immunohistochemistry was compared in two studies: study A, the liver was fixed for a maximum of 7 days and then embedded in paraffin and stored for approximately 18 months, while in study B, the liver was fixed in formalin for 7 years and then embedded in paraffin and stored for approximately 18 months until sectioning and immunostaining. PCNA immunostaining was successful in the liver sections of both studies, irrespective of the length of formalin fixation. Furthermore, the S phase labeling indices (LI) determined via PCNA and Tdr were comparable, although not identical, in the two studies. Therefore, use of PCNA immunohistochemistry should allow retrospective staining of rodent tissues for the assessment of cell proliferative activity in formalin-fixed organs from previously conducted toxicity and carcinogenicity studies.
Assuntos
Divisão Celular , Regeneração Hepática , Fígado/citologia , Proteínas Nucleares/análise , Timidina/metabolismo , Animais , Antígenos de Neoplasias/análise , Autoantígenos/análise , Autorradiografia , Hepatectomia , Imuno-Histoquímica/métodos , Fígado/fisiologia , Masculino , Índice Mitótico , Antígeno Nuclear de Célula em Proliferação , Ratos , Ratos Endogâmicos F344 , Estudos Retrospectivos , TrítioRESUMO
Hepatocellular neoplasms, including neoplastic nodules (NN), are the most commonly induced tumors resulting from chemical carcinogen evaluation. Our objective was to image neoplastic nodules using computed tomography. In a preliminary study using rats with diethylnitrosamine (DEN) induced tumors, lesions smaller than 1.5 cm were difficult to identify by CT. Since NN do not take up excess iron whereas normal liver does accumulate iron, we studied iron as a CT contrast material. Hemochromatosis was induced in 15 control rats by subcutaneous injections of iron dextran. A linear increase in iron-loading dose produces a linear CT liver enhancement (r = 0.97): 68, 80, 84, 94, and 104 HU at 0, one, two, four, and six weeks, respectively. No loss of enhancement was noted ten weeks later. Rat hepatic tissue was chemically analyzed after a similar iron-loading regimen. The iron concentration (microgram/g hepatic tissue) progressively increased during the first four weeks of loading and remained stable for three weeks following iron-loading. Four animals that had been given DEN and iron were examined by CT scanning to detect small NN. Iron-enhanced CT allowed the visualization of small lesions (less than 5 mm). Histopathologic sections confirmed a homogeneous pattern of iron uptake in normal liver with a deficiency of iron in NN. We conclude that CT scanning following iron-loading is a noninvasive method to detect small nodules and may provide a method to study the progression or regression of small liver nodules in rodents.
Assuntos
Dietilnitrosamina/toxicidade , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Nitrosaminas/toxicidade , Tomografia Computadorizada por Raios X/métodos , Animais , Hemocromatose/induzido quimicamente , Complexo Ferro-Dextran/administração & dosagem , Complexo Ferro-Dextran/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Intensificação de Imagem Radiográfica/métodos , Ratos , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Preliminary results from the National Toxicology Program (NTP) bioassays of furan given by gavage indicate the induction of hepatocellular carcinomas in male F-344 rats and in both sexes of B6C3F1 mice, and cholangiocarcinomas in both sexes of rats. To assess the genotoxicity of furan, chemically induced unscheduled DNA synthesis was evaluated in the in vivo hepatocyte DNA repair assay. Furan did not induce unscheduled DNA synthesis in hepatocytes isolated after single gavage treatment of male F-344 rats (5, 30, and 100 mg/kg) or male B6C3F1 mice (10, 50, 100, and 200 mg/kg). Furan induced cytotoxicity and enhanced cell proliferation were evaluated in livers of rats and mice as events that also might give rise to mutations and/or drive tumor formation. The labeling index (LI, percentage of hepatocyte nuclei in S-phase) was measured histoautoradiographically following a single gavage administration of furan (30 mg/kg, male rats; 50 mg/kg, male mice) followed by an injection of 3H-thymidine 2 hr prior to sacrifice. Hepatocellular necrosis and a sharp increase in LI (23.9 for mice and 17.8 for rats vs. less than 0.5 for controls) was observed 48 hr after treatment with furan, indicative of restorative cell proliferation secondary to cytotoxicity. Hepatocyte proliferation was evaluated also at the highest NTP bioassay dose (15 mg/kg/day for mice and 8 mg/kg/day for rats, 5 days/week) by labeling with 3H-thymidine administered via a 6 day osmotic pump implanted subcutaneously. Necrosis and inflammation were observed along the subcapsular visceral surface of the left or caudate liver lobes, likely due to diffusion of furan directly through the stomach to the liver. After 6 weeks of furan administration, male and female rats, but not mice, exhibited bile duct hyperplasia as well as metaplasia in the areas of fibrosis along the subcapsular visceral surface of the left or caudate liver lobes. The fold increase in hepatocyte LI in treated animals relative to the combined controls measured at weeks 1, 3, and 6 ranged from 39 to 5 for male mice, 18 to 51 for male rats, and 12 to 19 for female rats. Taken together, these data suggest that mechanisms other than direct DNA-reactivity might explain the profile of oncogene mutations observed in the mouse liver tumors, including selective promotion of different subpopulations of preneoplastic cells and/or mutational events secondary to sustained cell proliferation or inflammation. The extensive amount of furan-induced cell proliferation subsequent to cytotoxicity likely had a significant impact on tumor development, and such data should be considered in risk evaluations for this compound.
Assuntos
Furanos/toxicidade , Fígado/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Divisão Celular , Células Cultivadas , Reparo do DNA , Enzimas/sangue , Feminino , Furanos/administração & dosagem , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Ratos , Ratos Endogâmicos F344 , Fatores Sexuais , Fatores de TempoRESUMO
The ability of the peroxisome proliferator WY-14,643 to act as an initiator of hepatocarcinogenesis was examined using a modified growth-selection protocol in rats. Feeding rats 0.1% WY-14,643 in the diet for 3 weeks, coupled with partial hepatectomy after 10 days, failed to initiate the development of growth-selectable foci identifiable by 3 enzyme histochemical stains. Elevation of palmitoyl CoA oxidase activity in WY-14,643 rats from 1 to 11 days after partial hepatectomy suggested tht peroxisome proliferation, even when coupled with hepatocellular DNA replication, did not result in initiation. The failure of WY-14,643 to initiate growth-selectable foci may indicate that promotional activity is important in the hepatocarcinogenicity of the peroxisome proliferators.
Assuntos
Carcinógenos , Neoplasias Hepáticas/induzido quimicamente , Microcorpos/fisiologia , Pirimidinas/toxicidade , Animais , Hepatectomia , Peróxido de Hidrogênio , Neoplasias Hepáticas/ultraestrutura , Masculino , Microcorpos/ultraestrutura , Oxirredutases , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344RESUMO
Aniline and several structurally-related aromatic amines produce spleen tumours in rats given high doses of compound in 2-year bioassay studies. Evaluation of the pathogenesis of the splenic lesions and characterization of the disposition of radiolabelled aniline in animals suggests that the spleen tumours may be a secondary response resulting from chemically-mediated erythrocyte toxicity. It is proposed that compound-derived toxicity to erythrocytes results in scavenging of damaged red blood cells by the spleen, initiating a series of events which may contribute to the development of spleen tumours. These events potentially include (i) specific accumulation of the parent compound or toxic metabolite(s) carried to the spleen by erythrocytes; (ii) deposition of erythrocytic debris, particularly iron, which may catalyse tissue-damaging free-radical reactions; and (iii) induction of splenic hyperplasia resulting from erythrocyte overload. Linkage of the splenic tumorigenicity of these aromatic amines to an initial toxic event in the erythrocyte suggests that the carcinogenicity of such compounds may be determined by a definable threshold dose, i.e. the events leading to the carcinogenicity are not initiated until the capacity of the red blood cell to cope with the toxic insult is exceeded.
Assuntos
Compostos de Anilina/toxicidade , Esplenopatias/induzido quimicamente , Compostos de Anilina/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Hemossiderina/metabolismo , Masculino , Mutagênicos , Fatores Sexuais , Especificidade da Espécie , Neoplasias Esplênicas/induzido quimicamenteRESUMO
The effect of subchronic exposure to formaldehyde (HCHO; 6 ppm; 6 hr/day, 5 days/wk for 4 wk) on the HCHO concentration in the blood of three rhesus monkeys was investigated. Immediately after the final exposure, the monkeys were sedated, and blood samples were withdrawn 7 min after the end of exposure. The HCHO concentration in the blood, determined by gas chromatography-mass spectrometry was 1.84 +/- 0.15 micrograms/g blood and did not differ significantly after a further 45 hr without exposure to HCHO (2.04 +/- 0.40 micrograms/g blood). The average concentration of HCHO in the blood of exposed monkeys was also not significantly different from that of three unexposed controls (2.42 +/- 0.09 micrograms/g blood). However, individual monkeys differed significantly from one another with respect to their blood concentrations of HCHO. These results indicate that subchronic inhalation exposure of non-human primates to HCHO has no significant effect on the HCHO concentration in the blood, and that the average concentration of HCHO in the blood of monkeys is similar to that in the blood of humans.
Assuntos
Formaldeído/sangue , Administração por Inalação , Animais , Macaca mulattaRESUMO
Two known cholangiotoxic agents, alpha-naphthylisothiocyanate (ANIT) and p-phenylenediisothiocyanate (PDT), were administered in single doses to mice to study their effects on the gallbladder. Both compounds caused maximal bile duct necrosis and periportal hepatocytic necrosis at 24 hours. In contrast, the gallbladders were edematous but not necrotic at 24 hours after treatment. At 48 hours, and in some animals up to four days, severe cholecystitis was present, while bile ducts revealed progressive regeneration. The delay in the onset of gallbladder lesions was assumed to be the result of the toxic agent concentration in gallbladder bile after hepatic bile secretion was suppressed for 24 hours. The lesions provoked by PDT were similar to those induced by ANIT, except for a hemorrhagic component.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Colecistite/induzido quimicamente , Tiocianatos/toxicidade , Doença Aguda , Animais , Ductos Biliares Intra-Hepáticos/patologia , Ductos Biliares Intra-Hepáticos/fisiologia , Colecistite/patologia , Feminino , Vesícula Biliar/patologia , Fígado/patologia , Hepatopatias/patologia , Camundongos , Necrose , Regeneração , Tiocianatos/administração & dosagemRESUMO
Three rations, an 18.71% protein swine starter diet (control), a basal diet containing 83 micrograms of Cd/g of diet, or a basal diet containing 50% Chicago sewage sludge (CSS) providing 83 micrograms Cd/g of diet, were given to weanling pigs for 9 weeks. Depressed growth occurred in both groups given Cd-treated diets in comparison with growth in pigs fed the control ration. Microcytic, hypochromic anemia occurred in the group given the Cd-supplemented diet, but there were no significant differences in the hematologic values between pigs in the control and CSS-supplemented diets. Toxicosis probably resulted from combining CSS as 50% of the diet due to a deficiency of available protein or other essential nutrients or from the accumulation of hazardous chemical residues including Cd, Cu, Cr, Hg, Ni, or Zn. An increased amount of Fe in the CSS-treated ration apparently protected the pigs against the microcytic, hypochromic anemia.