Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene.
(41/1387)
1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity. (+info)
MAP kinase, a universal suppressor of sperm centrosomes during meiosis?
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We reported previously that inhibition of MAP kinase during meiosis in Urechis caupo eggs caused premature sperm aster formation and we reviewed indirect evidence that the suppression of sperm asters by MAPK during meiosis might be a universal mechanism (M. C. Gould and J. L. Stephano, 1999, Dev. Biol. 216, 348-358). We tested this proposition with oyster (Crassostrea gigas) and starfish (Asterina miniata) eggs, utilizing the MEK inhibitors U0126 and PD98059. Centrosomes, asters, and meiotic spindles were visualized by normal epifluorescence and confocal microscopy following indirect immunocytochemical staining for anti-beta-tubulin. When MAPK activation was inhibited, sperm asters in both species developed prematurely and tended to move toward the egg centrosomes, sometimes even fusing with the egg spindle or centrosomes. Meiotic spindles and polar body formation were also abnormal when MAPK was inhibited. (+info)
Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway.
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Disruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-beta1) has also been shown to trigger the reactivation of EBV in Burkitt lymphoma cell lines; however, the effect of TGF-beta1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-beta1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-beta1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-beta1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-beta1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests that TGF-beta1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-beta1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-beta1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-beta1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-beta1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-beta1 induces BZLF1 expression by an indirect mechanism. (+info)
Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts.
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In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors. (+info)
AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor.
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Growth factor modulation of estrogen receptor (ER) activity plays an important role in both normal estrogen physiology and the pathogenesis of breast cancer. Growth factors are known to stimulate the ligand-independent activity of ER through the activation of mitogen-activated protein kinase (MAPK) and the direct phosphorylation of ER. We found that the transcriptional activity of AIB1, a ligand-dependent ER coactivator and a gene amplified preferentially in ER-positive breast cancers, is enhanced by MAPK phosphorylation. We demonstrate that AIB1 is a phosphoprotein in vivo and can be phosphorylated in vitro by MAPK. Finally, we observed that MAPK activation of AIB1 stimulates the recruitment of p300 and associated histone acetyltransferase activity. These results suggest that the ability of growth factors to modulate estrogen action may be mediated through MAPK activation of the nuclear receptor coactivator AIB1. (+info)
Biomarkers of exposure to 1,3-butadiene as a basis for cancer risk assessment.
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1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being considerably more sensitive than rats. Urine metabolites are 1, 2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA). The reactive metabolite 1,2-epoxy-3-butene forms 1- and 2-hydroxy-3-butenyl valine adducts in hemoglobin (MHBVal). The objectives of the study were (1) to compare the suitability of MHBMA, DHBMA, and MHBVal as biomarkers for low levels of exposure to BD, and (2) to explore relative pathways of metabolism of BD in humans for comparison with mice and rats, which is important in relation to cancer risk assessment in man. Analytical methods of measuring MHBMA, DHBMA, and MHBVal were modified and applied in 2 studies to workers engaged in the manufacture and use of BD. Airborne BD concentrations were assessed by personal air monitoring. MHBMA in urine was more sensitive for monitoring recent exposures to BD when compared to DHBMA and could measure 8-h time weighted average exposures as low as 0.13 ppm. Relatively high natural background levels in urine restricted the sensitivity of DHBMA. The origin of this background is currently unknown. The measurement of MHBVal adducts in hemoglobin was a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm. Statistically significant relationships were found between urinary MHBMA and DHBMA concentrations, between either of these variables and 8-h airborne BD levels and between MHBVal adducts and average airborne BD levels over 60 days. The data on biomarkers demonstrated a much higher rate of hydrolytic metabolism of 1,2-epoxy-3-butene in humans compared to mice and rats, which was reflected in a much higher DHBMA/(DHBMA + MHBMA) ratio and in much lower levels of MHBVal in humans. Assuming a genotoxic mechanism, the data of this study, coupled with other published data on DNA and hemoglobin binding in mice and rats, suggest that the cancer risk for man from exposure to BD is expected to be less than for the rat and much less than for the mouse. (+info)
Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole.
(47/1387)
The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression. (+info)
Low [Mg(2+)](o) induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases.
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The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg(2+)](o)) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg(2+)](o) medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg(2+)](o), the stronger the contractions. The low-[Mg(2+)](o) medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC(50) levels obtained for these five antagonists are consistent with reported inhibitor constant (K(i)) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg(2+)](o) medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca(2+)](i)) peaks followed by a slow, sustained, and elevated plateau of [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries. Low-[Mg(2+)](o) medium induces rapid and stable increases in [Ca(2+)](i); these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg(2+)](o)-induced contractions or the elevation of [Ca(2+)](i). The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg(2+)](o)-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries. (+info)