Involvement of oxidative stress in bee venom-induced inhibition of Na+/glucose cotransporter in renal proximal tubule cells

1. The present study was conducted to examine the involvement of oxidative stress in bee venom-induced inhibition of the Na+/glucose cotransporter (alpha-methyl-d-glucopyranoside (alpha-MG) uptake), a typical functional marker of proximal tubules, in primary cultured rabbit renal proximal tubule cells (PTC). 2. Bee venom (> or = 1 microg/mL) increased lipid peroxide (LPO) formation over 30 min. The increase in [(3)H]-arachidonic acid (AA) release and LPO formation and the inhibition of alpha-MG uptake induced by bee venom (1 microg/mL) and melittin (a major component of bee venom; 0.5 microg/mL) were blocked by N-acetyl-l-cysteine, vitamin C and vitamin E, anti-oxidants. 3. Bee venom- and melittin-induced increases in LPO formation and inhibition of alpha-MG uptake were significantly prevented by mepacrine and AACOCF(3), phospholipase A(2) inhibitors. In addition, nordihydroguaiareic acid (a lipoxygenase inhibitor) and econazole (a cytochrome P-450 epoxygenase inhibitor), but not indomethacin (a cyclo-oxygenase inhibitor), prevented bee venom- and melittin-induced increases in LPO formation and inhibition of alpha-MG uptake. 4. Nordihydroguaiareic acid prevented bee venom- and melittin-induced increases in Ca(2+) uptake. Moreover, anti- oxidants significantly prevented bee venom- and melittin-induced increases in Ca(2+) uptake. 5. In conclusion, bee venom inhibits alpha-MG uptake via the phospholipase A(2)-oxidative stress-Ca(2+) signalling cascade in primary cultured rabbit renal proximal tubule cells.     

p-Anilinoaniline enhancement of dioxin-induced CYP1A1 transcription and aryl hydrocarbon receptor occupancy of CYP1A1 promoter: role of the cell cycle

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 μM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 μM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 μM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 μM pAA was cytostatic and induced a G(1) arrest that lasted ～7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.     

3',4'-dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells.

Treatment of MCF-7 and T47D human breast cancer cells with 3', 4'-dimethoxyflavone (3',4'-DMF) alone did not induce CYP1A1-dependent ethoxyresorufin O:-deethylase (EROD) activity or reporter gene activity in cells transfected with an aryl hydrocarbon (Ah)-responsive construct (pRNH11c). In contrast, 1 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced up to a 50- to 80-fold increase in EROD and reporter gene activity in MCF-7 and T47D cells. In cells cotreated with 1 nM TCDD plus 0.1-10 microM 3',4'-DMF, there was a concentration-dependent decrease in the TCDD-induced responses, with 100% inhibition observed at the 10 microM concentration. Gel mobility shift assays using rat liver cytosol and breast cancer cell nuclear extracts showed that 3',4'-DMF alone did not transform the AhR to its nuclear binding form, but inhibited TCDD-induced AhR transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF-7 and T47D cells. TCDD also inhibited estrogen-induced transactivation in MCF-7 cells, and this response was also blocked by 3',4'-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells.     

Effect of caffeic acid on apical transporters' dysfunction of renal proximal tubule cells under oxidative stress in vitro

The protective effect of caffeic acid (CA) against oxidative stress-induced inhibition of proximal tubule apical transporter was investigated. In the present study, 10 (-4) M H2O2 did not affect cell viability regardless of incubation time. However, it decreased apical transporters' activity such as Na (+)/glucose cotransporter, Na (+)/Pi cotransporter, and Na (+)/H(+) antiporter in the proximal tubule cells. CA (>10(-6) M) prevented H2O2-induced inhibition of apical transporters. Thus, we investigated its action mechanism. CA also prevented H2O2-induced lipid peroxides formation, arachidonic acid (AA) release, and Ca(2+) uptake. In conclusion, CA, in part, prevented H2O2-induced inhibition of apical transporter activity via decrease of AA release and Ca(2+) uptake in primary cultured renal proximal tubule cells.     

Possible aryl hydrocarbon receptor-independent pathway of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced antiproliferative response in human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ligand with high affinity for the aryl hydrocarbon receptor (AhR). It suppresses 17β-estradiol (E2)-induced cell proliferation in human breast cancer cells. Although it has been theorized that the AhR is involved in TCDD-induced antiestrogenic activity and antiproliferation in human breast cancer cells, some evidence suggests that these activities of chlorinated aromatic compounds also occur by AhR-independent pathways. Here, we investigated the possibility of TCDD-induced antiproliferative responses in human breast cancer cells through AhR-independent pathways. Compared with that in vehicle-treated controls, DNA synthesis was significantly suppressed in MCF-7 cells and ZR75-1 cells treated with TCDD at a very low concentration (0.01 nM), whereas that in human ovarian carcinoma OVCAR3 cells, human cervical carcinoma HeLa cells and human choriocarcinoma JEG-3 cells was unaffected, even by exposure to 10 nM TCDD. The suppression induced by TCDD was not associated with the estrogen receptor α-signaling pathway. Another AhR agonist, 3,3',4,4',5-pentachlorobiphenyl, had no effect on DNA synthesis in MCF-7 cells at concentrations high enough to induce the transactivation function of the AhR. Furthermore, in MCF-7 cells, knockdown of the AhR by RNA interference had no effect on TCDD-induced antiproliferation. These findings suggest that the principal pathways of TCDD-induced antiproliferation in breast cancer cells are not AhR dependent.     

Effects of calcium channel modulators on the proliferation of mouse spleen lymphocytes in vitro.

The effects of nimodipine (voltage-dependent calcium channel blocker), CGP 28392 and BAY K 8644 (novel dihydropyridine derivatives that are considered as calcium entry stimulators) on the spontaneous proliferation of mouse spleen lymphocytes were studied in vitro. [3H]-thymidine incorporation into DNA of lymphocytes was used as an sensitive index of the cell proliferation. It has been found that nimodipine (10(-4) M-10(-6) M) significantly inhibited the [3H]-thymidine uptake in a dose dependent fashion with ED50 value of 2.4 x 10(-5) M. Unexpectedly, CGP 28392 (10(-4) M-10(-7) M) acts as a calcium entry blocker and produces a strong inhibitory effect on lymphocyte proliferation (ED50-2 x 10(-5) M). BAY K 8644 at a high concentration (10(-4) M) also has an inhibitory effect but at a lower concentration (10(-6) M-10(-10) M) significantly increased [3H]-thymidine uptake and abolished the inhibitory effect of nimodipine. This effect of nimodipine was also reversed by 5 x 10(-3) M calcium chloride. These findings indicate that calcium channel modulators can regulate the proliferation of mouse spleen lymphocytes in vitro.     

The Jun N-terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor.

Exposure of the immortalized human breast epithelial cell line MCF10A to the Jun N-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) suppressed, in a concentration-dependent manner (IC50 is approximately 2 microM), the induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with SP600125 also suppressed the accumulation of TCDD-induced nuclear aryl hydrocarbon receptor (AhR)-DNA complexes, as assessed by electrophoretic mobility shift assays. Concentrations of SP600125 < or = 50 microM did not transform the AhR into a DNA-binding species when added to rat liver cytosol. However, addition of SP600125 to cytosol just before TCDD addition completely suppressed AhR transformation and DNA binding (IC50 approximately 7 microM). Sucrose gradient analyses using rat liver and murine hepatoma 1c1c7 extracts demonstrated that SP600125 competed with TCDD for binding to the AhR. These results suggest that SP600125 is an AhR ligand and functions as an AhR antagonist at concentrations used to pharmacologically inhibit JNK.     

Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway

1. It has been demonstrated that oxidized low-density lipoprotein (OX-LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX-LDL-induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with mitogen-activated protein kinase (MAPK) activation in rat cultured VSMCs. 2. Both native-LDL (N-LDL) and OX-LDL induced a time- and concentration-dependent incorporation of [(3)H]-thymidine in VSMCs. 3. OX-LDL induced time- and concentration-dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX-LDL-induced responses. 4. Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX-LDL. 5. Removal of Ca(2+) by BAPTA/AM or depletion of the internal Ca(2+) pool by thapsigargin significantly inhibited OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation. 6. OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration-dependent manner. 7. Overexpression of dominant negative mutants of Ras (H-Ras-15A) and Raf (Raf-N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. 8. These results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G protein-coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB in rat cultured VSMCs.     

Sustained aryl hydrocarbon receptor activity attenuates liver regeneration

In hepatocyte-derived cell lines, either loss of aryl hydrocarbon receptor (AhR) function or treatment with a persistent AhR agonist such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can disrupt G1 phase cell cycle progression. The present study used liver regeneration to explore mechanistically how AhR activity modulates hepatocyte proliferation in vivo. Treatment of mice with 20 mug/kg TCDD 1 day before 70% partial hepatectomy (PH) resulted in a 50 to 75% suppression in liver regeneration. Impaired proliferation was not associated with changes in levels of interleukin-6 or tumor necrosis factor-alpha, which prime quiescent hepatocytes to enter G1 phase. In fact, administration of TCDD 12 h after PH, a period well beyond the priming phase, still induced the G1 arrest. Decreased proliferation in TCDD-treated mice correlated with reduced cyclin-dependent kinase-2 (CDK2) activity, a pivotal regulator of G1/S phase transition. In contrast to observations made in cell culture, suppressed CDK2 activity was not strictly associated with increased binding of the CDK2 inhibitors p21Cip1 or p27Kip1. However, TCDD decreased levels of cyclin E binding to CDK2, despite normal cyclin E expression. The evidence also suggests that TCDD-induced hepatic growth arrest depends upon sustained AhR activity because transient AhR activation in response to endogenous queues failed to suppress the regenerative response. These findings establish a functional role for the AhR in regulating normal cell cycle control during liver regeneration.     

A comparative study on the effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin polychlorinated biphenyl126 and estrogen in human bronchial epithelial cells

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.     

Estradiol enhances and estriol inhibits the expression of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in a mouse ovarian cancer cell line

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous pollutant and promoter of carcinogenesis. This study investigated the interaction between TCDD and different estrogens in a cancer cell line (ID8) derived from mouse ovarian epithelium. TCDD-induced ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P4501A1 (CYP1A1) expression in a dose- and time-dependent manner. Estrogen receptor (ER) alpha mRNAs were constitutively expressed, but ER beta and progesterone receptor (PR) mRNAs were not expressed. Induction of EROD by TCDD was completely inhibited by a alpha-naphthoflavone and phenanthroline, two aryl hydrocarbon receptor (AhR) antagonists. Progesterone and gonadotropins (FSH and LH) had no effect on the induction of EROD by TCDD. Congeners of 17beta-estradiol (E2) increased the induction of EROD activity by TCDD dose-dependently in the relative potency order: estrone (El)>E2> or = 4-hydroxyestradiol (4OHE2)> or = 2-hydroxyestradiol (2OHE2). In contrast, estriol (E3) decreased EROD activity induced by TCDD. E2 increased TCDD-induced CYP1A1 protein and mRNA whereas E3 decreased both the protein and mRNA. E2 did not alter luciferase activity induced by TCDD in cells transfected with a luciferase reporter containing dioxin response elements (DRE) or a CYP1A1 promoter. In contrast, E3 dose-dependently decreased the luciferase activity. A pure anti-estrogen (ICI 182780) inhibited the interaction between E2 and TCDD but did not block E3's effect on EROD activity. These results indicate that E2 may affect TCDD-induced CYP1A1 expression by a mechanism different from E3 in ID8 cells. It appears that the potentiation of E2 in the induction of CYP1A1 by TCDD occurs by a mechanism involving ER alpha since a specific ER antagonist blocked the potentiation. The inhibitory effect of E3 may be due to a rapid direct effect on EROD and a later suppression of CYP1A1 expression.     

Antiproliferative and antiangiogenic effects of 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, in human umbilical vascular endothelial cells

There is increasing interest in the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and polycyclic aromatic hydrocarbons on cardiovascular diseases. Their chemical structures are similar, although polycyclic aromatic hydrocarbons contain no chlorine as does TCDD. The biochemical mechanism of their action is mainly mediated by the aryl hydrocarbon receptor. In addition, oxidative stress also plays a role in the biological and toxic effects of these chemicals. In this study, we used an aryl hydrocarbon receptor agonist, 3-methylcholanthrene (3-MC), to investigate its effect on the proliferation and angiogenesis of human umbilical vascular endothelial cells. 3-MC suppressed DNA synthesis of human umbilical vascular endothelial cells as determined by [(3)H]thymidine incorporation in a concentration-dependent fashion and arrested cells at the G0/G1 phase of the cell cycle. Interestingly, the inhibition of DNA synthesis by 3-MC was eliminated to a greater extent by aryl hydrocarbon receptor antagonists, alpha-NF (0.5 and 1 microM) and resveratrol (5 and 10 microM), than by the antioxidant, N-acetylcysteine (5 and 10 mM). Cell permeability, adhesion, and tube formation in human umbilical vascular endothelial cells exposed to 3-MC decreased in concentration-dependent manners. We also demonstrated that cell adhesion signaling (phosphorylated focal adhesion kinase (FAK)) decreased upon 3-MC treatment, suggesting that cell adhesion inhibited by 3-MC might be due to inhibition of cell adhesion signaling. Additionally, alpha-naphthoflavon (alpha-NF) ameliorated the effects of 3-MC on cell permeability, adhesion and tube formation, indicating the involvement of the aryl hydrocarbon receptor in angiogenesis. The results suggest that the adverse effects of 3-MC are mainly mediated by the aryl hydrocarbon receptor and not via increased oxidative stress.     

Cytotoxic and immunomodulative effects of flunitrazepam, zipeprol and trihexyphenidyl on human peripheral blood lymphocytes

The cytotoxic effects of flunitrazepam, zipeprol and trihexyphenidyl on cell viability of peripheral blood mononuclear cells from healthy volunteers were studied at concentrations from 10(-2) to 10(-8) M by 2 colorimetric in vitro assays: the neutral red uptake assay and thiazolyl blue tetrazolium bromide assay. All tested drugs of abuse were non-cytotoxic at concentrations lower than 10(-5) M. Possible immunomodulative effects of these substances were evaluated through phytohemagglutinin induced lymphocyte proliferation ([3H]-thymidine DNA incorporation assay) and by a 51Cr release natural killer assay. The 3 drugs studied produced statistically significant immunomodulative alterations on both immunological parameters.