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Archives of Toxicology (v.71, #10)

Control of the mutagenicity of aromatic amines by protein kinases and phosphatases I. The protein phosphatase inhibitors okadaic acid and ortho -vanadate drastically reduce the mutagenicity of aromatic amines by B. Oesch-Bartlomowicz; H. J. Arens; B. Richter; J. G. Hengstler; F. Oesch (pp. 601-611).

The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10⁻⁹–10⁻⁶ M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by >80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of ³²P-labelled phosphate from intracellularly prelabelled [³²P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.

Keywords: Key words Aromatic amines; Okadaic acid; Orthovanadate; Protein phosphatases; Mutagenicity

Cisplatin-induced apoptosis of immortalized mouse proximal tubule cells is mediated by interleukin-1β converting enzyme (ICE) family of proteases but inhibited by overexpression of Bcl-2 by Michio Takeda; Mami Kobayashi; Isao Shirato; Takako Osaki; Hitoshi Endou (pp. 612-621).

Cisplatin is known to induce serious renal damage including acute renal failure, the major site of renal injury appears to be localized to the third segment of the proximal tubule (S₃). Apoptosis occurs during a variety of acute injuries to tubule cell. The purpose of this study was to determine whether cisplatin induces apoptosis of immortalized mouse S₃ cells, and to define the intracellular pathways leading to cell death. S₃ cells exposed to cisplatin exhibited biochemical, morphological, and flow cytometric changes characteristic of apoptosis associated with slight necrosis. Cisplatin-induced apoptosis could be inhibited by overexpression of crmA, a cowpox virus gene, of which the product is known to suppress activities of the interleukin-1β converting enzyme (ICE) family proteases. On the other hand, overexpression of bcl-2, an antiapoptotic oncogene, rendered S₃ cells partially resistant to cisplatin. These results indicate that cisplatin-induced proximal tubule damage is associated with apoptosis, which is positively modulated by the ICE family of proteases and negatively by the product of bcl-2.

Keywords: Key words Cisplatin; Apoptosis; Proximal tubule; ICE

Relationship of anesthetic activity of alkyl acetates to hydrophobicity and in vivo effect on membrane fluidity in mice by Hideji Tanii; Xiao-Ping Zhang; Rie Oka; Kiyofumi Saijoh (pp. 622-626).

In vivo anesthetic activity of alkyl acetates in mice was studied in relation to hydrophobicity and the in vivo effect on membrane fluidity. The anesthetic potency (AD₅₀) of alkyl acetates was determined; AD₅₀ shows the i.p. dose required to anesthetize 50% of mice from the treated group. We used log P (n-octanol/water partition coefficient) as an operational definition of hydrophobicity. Membrane fluidity was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probe. Log (1/AD₅₀) was a parabolic function of log P, and the value of log P that corresponds to the minimum AD₅₀ was estimated to be 2.08. Brain synaptosomal membranes were prepared from mice 30 min after dosing with each of the three alkyl acetates applied at 1.5-fold AD₅₀: n-butyl, n-amyl, and n-hexyl acetate. In each alkyl acetate group, most of the animals were anesthetized (>68%). Decreased membrane fluidity was observed for the animals that were anesthetized while no change in the fluidity was seen for the animals that were not anesthetized. The results suggest an involvement of decreased DPH fluidity in alkyl acetate-induced anesthesia.

Keywords: Key wordsn-Butyl acetate; n-Amyl acetate; Anesthesia; Hydrophobicity; Membrane fluidity

Effect of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of adhesion molecules on human neutrophils by Kaisa M. Heiskanen; Silvia Münzing; Fritz Krombach; Kai M. Savolainen (pp. 627-632).

The effects of linoleic acid, linoleic acid anilide, and arachidonic acid on the expression of CD11b/CD18, CD11c/CD18 integrins and l-selectin on human neutrophils were studied by flow cytometry in a whole blood assay. None of these compounds had any effect on the basal expression of CD11b, CD11c, or l-selectin in the concentration range of 20–100 μM. However, linoleic acid at a concentration of 1000 μM slightly up-regulated CD11b and CD11c by a factor of 2.1 and 1.7, respectively. Linoleic acid, linoleic acid anilide, and arachidonic acid did not affect the formyl-methionyl-leucyl-phenylalanine induced up-regulation of CD11b or CD11c. However, linoleic acid and linoleic acid anilide slightly inhibited the phorbol myristate acetate (PMA)-induced expression of CD11b, which was decreased by 27 and 21% at concentrations of 100 and 1000 μM, respectively. Likewise, arachidonic acid at 40 μM inhibited the PMA-induced expression of CD11b by 19%. Our results suggest that linoleic acid, linoleic acid anilide, and arachidonic acid do not dramatically affect the expression of leukocyte adhesion molecules in a whole blood assay.

Keywords: Key words Linoleic acid; Linoleic acid anilide; Arachidonic acid; Toxic Oil Syndrome; Adhesion molecules

Inhibitory effect of nicotinamide to paraquat toxicity and the reaction site on complex I by Tetsuhito Fukushima; Tongqiang Gao; Toshinaga Tawara; Nobumasa Hojo; Akio Isobe; Yosuke Yamane (pp. 633-637).

The inhibitory effect and mechanism of action of nicotinamide to paraquat toxicity were studied in male Sprague-Dawley rats. Proteins of submitochondrial particles (SMP), especially of mol. wt. 25–30 kDa, in rat lungs were destroyed by paraquat radicals, and aggregated protein bands ∼100 kDa were observed by polyacrylamide electrophoresis. The competitive inhibition effects were observed of nicotinamide on NADH oxidation by paraquat via SMP in rat lungs and the K _i was 9.3 mM. The inhibitory effects of nicotinamide on lipid peroxidation by paraquat with rat lung and liver SMP were verified. The times of occurrence of dyspnea and death in rats after paraquat exposure were delayed by nicotinamide administration. The activity of NADH: ubiquinone reaction of NADH:ubiquinone oxidoreductase (complex I) in rat lung was reduced 24 h after paraquat exposure, and was protected by nicotinamide. The activity of NADH:ferricyanide reaction of complex I was, however, reduced by administration not only of paraquat but also nicotinamide. These results imply that nicotinamide is inhibitory to paraquat toxicity. Nicotinamide, paraquat, and ferricyanide may react at overlapping sites on complex I.

Keywords: Key words Paraquat toxicity; Mitochondria; Complex I; Nicotinamide; Inhibitory effect

Time courses of hepatic injuries induced by chloroform and by carbon tetrachloride: comparison of biochemical and histopathological changes by P.-Y. Wang; T. Kaneko; H. Tsukada; M. Nakano; T. Nakajima; A. Sato (pp. 638-645).

The relationship was investigated between biochemical and morphological changes in chloroform (CHCl₃)- and carbon tetrachloride (CCl₄)-induced liver damage. The time courses of hepatic microsomal cytochrome P450 (CYP) content, hepatic microsomal CYP2E1 activity, hepatic reduced glutathione (GSH) content, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were examined in relation to the liver morphology in rats orally treated with CHCl₃ or CCl₄ (3.35 mmol/kg). The CYP content and the activity of CYP2E1 markedly decreased in the CCl₄-treated rats 3 h after treatment compared to much lower decreases in the CHCl₃-treated rats. The hepatic GSH content was decreased to a similar extent in both groups of rats at 3 h after treatment; in the CCl₄-treated rats, the GSH content continued to decrease, reaching a minimum at 24 h and without attaining the normal level at 72 h after treatment. By contrast, hepatic GSH content in the CHCl₃-treated rats began to increase from 6 h, attaining complete recovery 48 h after treatment. Plasma ALT and AST activities were significantly elevated by CCl₄ as early as 3 h after treatment, while the activities in the CHCl₃-treated rats did not increase until 6 h after treatment. In both groups of rats, ALT and AST activities reached a maximum at 24 h, and gradually decreased, remaining at abnormal levels at 72 h. Hepatic cells in the CCl₄-treated rats were found to be necrotic as early as 3 h post-treatment, whereas few or no morphological changes appeared in the liver of CHCl₃-treated rats. The extent of necrosis was at a maximum 24 h after treatment in both CHCl₃- and CCl₄-treated rats. In addition, some necrotic cells remained in the liver of CCl₄-treated rats 72 h after treatment, while the necrosis in the CHCl₃-treated rats was almost negligible. The present results indicate that almost the same time-courses of biochemical and morphological changes were followed in rats of both the CHCl₃- and CCl₄-treated groups.

Keywords: Key words Chloroform; Carbon tetrachloride; Liver; Cytochrome P450; CYP2E1

Effect of phenobarbital on hepatic eicosanoid concentrations in rats by Rhonda S. Peebles; Howard P. Glauert (pp. 646-650).

Phenobarbital is an efficacious tumor-promoting agent in the liver. Studies using inhibitors of eicosanoid synthesis have suggested that eicosanoids are important in the promotion of hepatocarcinogenesis by phenobarbital. We therefore hypothesized that hepatic eicosanoid concentrations are altered following phenobarbital administration. Male Sprague-Dawley rats were fed one of four levels of phenobarbital (0, 0.02, 0.05, and 0.1%). Eight rats from each of the four groups were killed after 10, 24, and 44 days for determination of liver weight and for preparation of microsomes. No significant difference was found among rat weights; however, liver weights were significantly higher in rats fed phenobarbital. Assay of benzyloxyresorufin-O-dealkylase activity showed that cytochromes P-450 2B1 and 2B2 were induced in response to phenobarbital administration. Prostaglandin E₂ concentrations were found to be significantly decreased by phenobarbital treatment after 10 and 24 days, but not after 44 days. Prostaglandin F_2α levels were decreased only by the lowest dietary phenobarbital concentration. Hepatic concentrations of leukotriene C₄ were decreased significantly at 10 days and at 44 days (only for the group administered the highest percentage concentration of phenobarbital), but not at 24 days. These results show that the investigated eicosanoids are generally slightly decreased by phenobarbital administration. Elevated eicosanoid levels therefore do not appear to be necessary for the promoting activity of phenobarbital.

Keywords: Key wordsPhenobarbital; Prostaglandin; Leukotriene; Hepatocarcinogenesis

Pyruvate alleviates toxic effects of ethanol on cells in culture by K. Fukaya; M. Miyazaki; H. Pu; B. Katayama; Y. Inoue; R. Ohashi; C. Nakamura; M. Namba (pp. 651-654).

Cytotoxic effects of ethanol on cultured human hepatocytes and fibroblasts differed with the type of culture medium used. When cytotoxic effects of ethanol were compared between culture systems using either RPMI-1640 or Dulbecco's modified Eagle's medium (DMEM), the cytotoxicity was more prominent with the former medium than with the latter. A reduction of the cytotoxic effects appeared to be due to the pyruvate contained in DMEM. The protective effect of pyruvate against ethanol toxicity may be related to tricarboxylic acid (TCA) cycle activity because addition of malate to the medium also reduced the cytotoxic effects. Our results suggest that drug cytotoxicity testing in vitro must be done using various types of culture medium.

Keywords: Key words Toxicity test; In vitro; Ethanol; Pyruvate; TCA cycle

Control of the mutagenicity of arylamines by protein kinases and phosphatases: II. Lack of response of rat liver N-acetyl transferases to phosphorylation modulators by Palghat Raghunathan Padma; Barbara Oesch-Bartlomowicz; Jan Georg Hengstler; Franz Oesch (pp. 655-659).

Treatment of rat hepatocytes with the phosphatase inhibitors okadaic acid or ortho-vanadate had led to an 80% decrease in the bacterial mutagenicity of several aromatic amines metabolically activated by these hepatocytes. This is the most dramatic change yet demonstrated in mutagenicity by phosphorylation modulation. However, incorporation of phosphate into and catalytic activity of cytochromes P450 (CYP) 1A1 and 1A2, the major catalysts for the first step in the toxication of aromatic amines, were unchanged. We therefore investigated whether changes in the phosphorylation status would influence the activities of the N-acetyltransferases NAT1 and/or NAT2, being responsible for one of the two major pathways leading to the ultimate mutagens, the reactive esters which are derived from the N-hydroxylated metabolites of aromatic amines. Hepatocytes were derived from the livers of rats pretreated with CYP1A1/1A2 inducers and from untreated rats using conditions under which the phosphorylation-dependent drastic decrease of the arylamine mutagenicity was observed. Treatments were exposure to 1 mM dibutyryl-cAMP (protein kinase A stimulator), 100 nM okadaic acid or 20 nM calyculin A (preferential inhibitors of serine/threonine phosphatases PP2A and PP1, respectively), 2 mM ortho-vanadate (inhibitor of tyrosine phosphatases), and 50 mM NaF (stimulator of adenylate cyclase and non-specific inhibitor of protein phosphatases). None of the phosphorylation modulators led to a significant change in NAT1 or NAT2 activities. This was true for hepatocytes from rats which had been pretreated with inducers for CYP1A1 and CYP1A2 as well as from untreated rats. The inducers led to the expected increases in CYP1A1 and CYP1A2 but the NAT1 and NAT2 activities remained unchanged. Our study shows that the N-acetyl transferases NAT1 or NAT2, the catalysts responsible for the formation of the highly reactive N-acetoxy derivatives of N-hydroxylated aromatic amines, are not responsible for the drastic decrease in arylamine genotoxicity after treatment of the metabolizing system with protein phosphatase inhibitors. The data also shown that NAT1 and NAT2 are not regulated by the classical xenobiotic metabolizing enzyme inducers nor by any of the phosphorylation modulators used.

Keywords: Key words N-acetyl transferases; Phosphorylation; Protein phosphatases; Protein kinase A

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Archives of Toxicology (v.71, #10)