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Archives of Toxicology (v.72, #7)
Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver by Nobumitsu Hanioka; Hideto Jinno; Tetsuji Nishimura; Masanori Ando (pp. 387-394).
We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The K m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The V max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA.
Keywords: Key words Bisphenol A; Cytochrome P450; Cytochrome P450 isoforms; Rat liver microsomes; Endocrine disruptor
Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene by Ruei-Ming Chen; Ming W. Chou; Tzuu-Huei Ueng (pp. 395-401).
The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days. Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels. Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102. The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes. Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1–12–3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung. RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA. 6-NC had no effect on the kidney P450 protein and mRNA. The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.
Keywords: Key words 6-Nitrochrysene; Cytochrome P450; Mutagenicity; Nitroreductase
Induction of ethoxyresorufin O-deethylase (EROD) and endothelial activation of the heterocyclic amine Trp-P-1 in bird embryo hearts by Anita Annas; Björn Brunström; Ingvar Brandt; Eva B. Brittebo (pp. 402-410).
The xenobiotic-metabolizing activity of avian heart was investigated in chicken and Eider duck embryos exposed to aryl hydrocarbon (Ah) receptor agonists in ovo. Both β-naphthoflavone (BNF) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) induced 7-ethoxyresorufin O-deethylase (EROD) activities in chicken embryo hearts whereas Eider duck embryos only responded to BNF. The differential responses of chicken and Eider duck embryos were used to examine the involvement of Ah receptor-mediated enzyme induction in the activation of the environmental and food mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1). As determined by light microscopic autoradiography, there was a highly selective binding of non-extractable 3H-Trp-P-1-derived radioactivity in endothelial cells of large vessels and capillaries in hearts of BNF- and PCB 126-treated chicken embryos. No binding occurred at these sites in vehicle-treated controls. There was also a strong endothelial binding of 3H-Trp-P-1 in hearts of BNF-treated Eider duck embryos whereas no binding occurred in hearts of PCB 126-treated Eider duck embryos. A positive correlation between induction of EROD activity and covalent binding of 3H-Trp-P-1 to protein in heart homogenates from BNF- and PCB 126-treated chicken and Eider duck embryos was also observed. The results suggest a cytochrome P450 1A (CYP1A)-mediated activation of Trp-P-1 in avian heart endothelial cells although involvement of other Ah receptor-regulated enzymes is also possible. We propose that heart endothelial cells may be targets for bioactivation and toxicity of environmental contaminants in birds exposed to Ah receptor agonists.
Keywords: Key words 3-Amino-1; 4-dimethyl-5H-pyrido[4; 3-b] indole (Trp-P-1); Endothelial cells; Heart; CYP1A; EROD
Effects of fluoroquinolones and magnesium deficiency in murine limb bud cultures by Christian Förster; Marcus Rücker; Mehdi Shakibaei; Irmela Baumann-Wilschke; Jürgen Vormann; Ralf Stahlmann (pp. 411-419).
Quinolone-induced arthropathy is probably caused by a lack of functionally available magnesium in immature joint cartilage. We used an in vitro assay to study the effects of fluoroquinolones on cartilage formation in mouse limb buds from 12-day-old mouse embryos in regular and in magnesium-deficient medium. Omission of magnesium from the medium had no adverse effect on the outcome of the culture: limb buds grew and differentiated well in regular and in magnesium-deficient Bigger's medium. Lack of calcium, however, severely impaired the development of the explants; this result was even more enhanced when both minerals (magnesium and calcium) were omitted. Electron microscopy revealed cell necrosis and deposition of electron-dense material in the vicinity of chondrocytes from limb buds after 6 days in a magnesium-free medium. A series of seven fluoroquinolones was tested at 30, 60, and 100 mg/l medium. At a concentration of 30 mg/l sparfloxacin only had a slight effect on limb development. At concentrations of 60 and 100 mg/l sparfloxacin, temafloxacin and ciprofloxacin impaired limb development in␣vitro concentration-dependently. The effects were enhanced in a magnesium-deficient medium (concentration of magnesium <10 μmol/l). Fleroxacin, lomefloxacin and ofloxacin impaired limb development only slightly; no significant differences were recognizable between the outcome in regular and in magnesium-deficient medium. Pefloxacin did not show any effect on limb development in both media. Using electron microscopy, very similar alterations as described above for the limbs cultured in magnesium-deficient medium were observed with ofloxacin at a concentration of 30 mg/l, which had no effect on the growth of the explants when evaluated macroscopically. The affinity of six fluoroquinolones to magnesium was determined by the use of a fluorescence assay. The affinity to magnesium correlated with the activity of the drugs in the limb bud assay. We conclude that fluoroquinolones have no effect on murine limb development in vitro at concentrations that are achieved under therapeutic conditions (peak concentrations approx. 1–5 mg/l in plasma). Effects at higher concentrations (60 and 100 mg/l) are slightly enhanced (factor 2) if the magnesium concentration in the medium is low. Macroscopically, limbs develop regularly in a magnesium-free medium, but ultrastructurally typical alterations are exhibited (e.g. cell necrosis and pericellular deposition of electron-dense material).
Keywords: Key words Fluoroquinolones; Chondrotoxicity; Magnesium; Calcium; Limb bud culture
The role of glutathione S-transferase- and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat by M. P. Chamberlain; E. A. Lock; B. A. Gaskell; C. J. Reed (pp. 420-428).
The aim of this study was to investigate the role of metabolic activation in the olfactory toxicity of methyl iodide (MeI). Adult male rats were exposed via nose-only inhalation to 100ppm MeI for 0–6h, and non-protein sulphydryl (NP-SH) concentrations determined in selected tissues. Depletion of NP-SH occurred in all tissues, but was most marked and rapid in the respiratory epithelium of the nasal cavity and the kidney. Olfactory, lung and liver NP-SH levels were affected to a lesser extent, and those of the brain declined by only 20–30% over the whole time course. In order to modulate glutathione (GSH) status, animals were pre-treated with (1) phorone plus l-buthionine sulphoximine (BSO), which depleted NP-SH levels in all the tissues examined, or (2) the isopropyl ester of GSH (IP-GSH), which was shown to replenish NP-SH concentrations in all tissues except the liver of animals previously administered phorone. When animals were pre-treated with phorone plus BSO and then exposed to 100ppm MeI for 2h, there was a potentiation of the toxicity of MeI as judged by the clinical observations on the animals. In contrast, treatment with IP-GSH prior to and during exposure to MeI for 4h afforded a marked protection to the olfactory epithelium. In order to inhibit cytochromes P450, animals were pre-treated with cobalt protoporphyrin IX. This decreased hepatic cytochrome P450 concentrations by >90%, but when animals were then exposed to 100ppm MeI for 4h there was no effect on the severity of the olfactory lesion. These results indicate that conjugation of MeI with GSH is a detoxification rather than an activation pathway. Also, there is no major role for cytochrome P450-dependent oxidation in the development of the olfactory lesion.
Keywords: Key words Methyl iodide; Olfactory toxicity; Glutathione; Cytochromes P450; Glutathione S-transferases
Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile by John Whysner; Robert E. Steward 3rd; Di Chen; Carson C. Conaway; Lynne K. Verna; John P. Richie Jr.; Nusrat Ali; Gary M. Williams (pp. 429-438).
Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.
Keywords: Key words 8-Oxodeoxyguanosine; 8-Hydroxydeoxyguanosine; Epigenetic mechanisms; Methylnitrosourea; Oxidative DNA damage
The rodent nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin inhibits intercellular communication in rat but not guinea-pig hepatocytes, perturbing S-phase but not apoptosis by Fiona J. Elcock; J. Kevin Chipman; Ruth A. Roberts (pp. 439-444).
Previously, we have shown that the peroxisome proliferator (PP), nafenopin, induces S-phase in␣rat hepatocytes and suppresses apoptosis in hepatocytes from both rat and guinea-pig. Here, we confirm and extend these findings by defining the time course of␣growth perturbation and by correlating this with species differences in loss of gap junctional intercellular communication (GJIC). GJIC is associated with nongenotoxic carcinogenesis, possibly reflecting a tumour suppresser role of the connexins. Fluorescence microscopy of Hoechst 33258-stained rat or guinea-pig hepatocyte monolayers showed 1% apoptosis during the first 8 h of culture, peaking to 2–2.5% at 20–24 h. Nafenopin suppressed apoptosis compared with controls in both rat and guinea-pig, measured at 20 h and 24 h onwards, respectively. The induction of S-phase in rat hepatocytes by nafenopin could be detected as early as 4 h after compound addition whereas S-phase was not altered by nafenopin in guinea-pig hepatocytes. Intercellular communication as measured by intercellular transfer of microinjected Lucifer Yellow CH was observed during the first 14 h of primary rat hepatocyte culture peaking at a maximum value of 88 ± 3.0% after 7 h. In hepatocyte cultures from guinea-pig, dye-coupling levels were maintained between 88 ± 3.0 and 93 ± 3.0% within 2–10 h of culture and by 12 h showed only a slight decrease to 72 ± 3.0%. In the rat, significant inhibition was observed at 4 h after administration of nafenopin since GJIC was reduced by 20 ± 5% compared with vehicle control. By contrast, in the presence of nafenopin, the level of dye-coupling between guinea-pig hepatocytes did not decrease but remained between 85 ± 5 and 93 ± 3.0%, similar to that observed in control guinea-pig cultures. The data obtained contribute to our understanding of the role of GJIC inhibition in the perturbation of cell survival and proliferation caused by nongenotoxic hepatocarcinogens.
Keywords: Key words Apoptosis; S-phase; Gap junctional intercellular communication; Peroxisome proliferators; Nongenotoxic carcinogenesis
Chronic exposure to ozone and nitric acid vapor results in increased levels of rat pulmonary putrescine by Ram K. Sindhu; William J. Mautz; Yutaka Kikkawa (pp. 445-449).
In the past decade, there has been growing public concern for the human health effects of exposure to environmental pollutants. Ozone (O3) is one of the most reactive components of photochemical air pollution. Despite extensive investigations by many laboratories on the functional, biochemical, and cellular effects of O3 exposure in humans, animals, and in vitro systems, questions remain concerning the potential adverse effects to human health represented by chronic near-ambient exposure to this environmental pollutant. In the present investigation, the influence of inhalation of O3 and nitric acid (HNO3) vapor on polyamine levels was examined in rat lungs. Male F344/N rats were exposed nose-only to 0.15 ppm O3 and 50 μg/m3 HNO3 vapor alone and in combination for 4 hours/day, 3 days/week for a total of 40 weeks. At this time the animals were sacrificed and their lungs were examined for polyamine contents. Exposure to O3 and O3 plus HNO3 vapor caused a significant increase in the putrescine content of the lung compared to the air-exposed controls (P < 0.05). The concentrations of pulmonary spermidine and spermine were not significantly increased by exposure to either O3 or HNO3 vapor alone or in combination compared to the air-exposed controls. The role of polyamines in repair and anti-inflammatory processes has been␣discussed.
Keywords: Key words Ozone; Polyamine metabolism; Rat lung; Putrescine; Repair
Dietary Mg and/or K restriction enhances paraquat toxicity in rats by Kayoko Minakata; Osamu Suzuki; Shin-ichi Saito; Naoko Harada (pp. 450-453).
Effect of mineral restriction was studied to clarify which mineral in the diet is most indispensable in preventing paraquat (PQ) toxicosis. ODS rats were chosen as the experimental animal owing to the inability to synthesize vitamin C similarly to humans. Rats were fed with either mineral-adequate or restricted diets dosed with 125 ppm PQ. The mineral-adequate diet was based on the American Institute of Nutrition-76, and the restricted diet was one-half the amounts. Measurements were made on the onset day of PQ toxicosis, body weight changes during the feeding experiment, and changes of two acute phase reactant proteins – cysteine proteinase inhibitor and α1-proteinase inhibitor. The minerals tested were divided into three classes: I, largely needed, Ca, K, Na, and Mg; II, moderately needed, Mn, Fe, Zn, and Cu; and III, minutely needed, Cr and Se, respectively. Rats fed with a Mg-restricted diet showed a severe toxicosis but those with a K-restricted diet, a mild toxicosis. No appreciable effect was observed by restriction of other minerals. A synergistic effect was observed in the restriction of Mg and K.
Keywords: Key words Magnesium deficiency; Potassium deficiency; Paraquat; Rats; Cysteine proteinase inhibitor
GSTT1 null genotype frequency in a Turkish population by Basbug Oke; Fahri Akbas; Muge Aydin; Hakan Berkkan (pp. 454-455).
In humans, glutathione-S-transferases (GSTs) are involved in the detoxification of xenobiotics. A deletion polymorphism in the glutathione S-transferase Theta 1 gene (GSTT1 null genotype) is associated with an increased risk of certain forms of cancer. The distribution of this polymorphism in 240 healthy individuals has been examined, using a polymerase chain reaction (PCR)-based method. The GSTT1 null genotype frequency was 20% in a Turkish Population.
Keywords: Key words Glutathione S-transferases (GSTs); GSTT1; Polymerase chain reaction (PCR)
Glutathione transferase T1 and M1 genotype polymorphism in the normal population of Shanghai by Jianhua Shen; Goufang Lin; Wuxing Yuan; Jingwei Tan; Hermann M. Bolt; Ricarda Thier (pp. 456-458).
Glutathione transferases are known to be important enzymes in the metabolism of xenobiotics. In humans genetic polymorphisms have been reported for the hGSTM1 and hGSTT1 genes leading to individual differences in susceptibility towards toxic effects, such as cancer. This study describes the distribution of the two polymorphisms of hGSTT1 and hGSTM1 in the normal Chinese population of Shanghai. Out of 219 healthy individuals having been genotyped for GSTT1 and GSTM1, 108 (49%) were identified to be homozygously deficient for the GSTT1 gene and 107 (49%) for the GSTM1 gene.
Keywords: Key words Glutathione transferase; GSTT1; GSTM1; Polymorphism