FIA/MS analysis of temporally ozonated benzo[a]pyrene and pyrene and their reaction products: inhibition of gap junction-mediated intercellular communication.

In this study, the polycyclic aromatic hydrocarbons, benzo[a]pyrene (BaP) and pyrene, were subjected to temporal ozonation. The products from ozonation of 5 mg l(-1) BaP and 5 mg l(-1) pyrene, for varying time intervals (0, 1, 2, 3, 4, 5, 6, 8, 10, 20, and 30 min) were tested for their effects on gap junction-mediated intercellular communication (GJIC) in Clone 9 rat liver cells. Additionally, the ozonation products were also analyzed by flow injection analysis/mass spectrometry (FIA/MS) and the results were compared with the toxicity observed in the GJIC assay. Treatment of the Clone 9 cells with 5 mg l(-1) of ozonated BaP products resulted in a decrease in GJIC that was inversely proportional to the length of ozonation. The products from 1 min of ozonation resulted in a 92% decrease in the rate of GJIC, but with >5 min ozonation, the products did not suppress GJIC. In contrast, pyrene (0.5 mg l(-1)) required >10 min of ozonation to alleviate its effects on GJIC. FIA/MS, using atmospheric pressure chemical ionization (APCI), demonstrated products with higher molecular weights (MW) than their corresponding parent compounds, BaP (MW 252) and pyrene (MW 202). Ozonation of pyrene formed significantly fewer products than BaP. More importantly, pyrene ozonation products were constant from 1 to 10 min, while BaP ozonation products seemed to vary between time intervals. With the longer ozonation times (20 and 30 min), BaP and pyrene formed similar products (m/z peaks 157, 111, and 96). The suppression of GJIC by ozonated products seemed to correlate with oxidation of the aromatic ring framework. Further oxidation (longer ozonation times) to lower MW products correlated with restoration of normal GJIC.

Novel genomic targets in oxidant-induced vascular injury.

To study the complex interaction between oxidative injury and the pathogenesis of vascular disease, vascular gene expression was examined in male Sprague-Dawley rats given 35 or 70 mg/kg allylamine, a synthetic amine converted to acrolein and hydrogen peroxide within the vascular wall. Vascular lesions and extensive vascular remodeling, coupled to increased production of 8-epi-PGF2alpha, nuclear localization of NFkappaB, and alterations in glutathione homeostasis, were observed in animals treated with allylamine for up to 20 days. Transcriptional profiling, immunohistochemistry, and in situ hybridization showed that genes involved in adhesion and extracellular matrix (ECM) (alpha(1) integrin, collagen), cytoskeletal rearrangements (alpha-smooth muscle actin, alpha-tropomyosin), and signal transduction (NFkappaB, osteopontin, and LINE) were altered by oxidant treatment. To evaluate mechanisms of gene dysregulation, cultured aortic smooth muscle cells were challenged with allylamine or its metabolites and processed for molecular analysis. These agents increased formation of reactive oxygen species and elicited changes in gene expression similar to those observed in vivo. Oxidative stress and changes in gene expression were inhibited by N-acetyl cysteine, a precursor of glutathione. These results indicate that genes along the ECM-integrin-cytoskeletal axis, in addition to LINE, are molecular targets in oxidant-induced vascular injury.

The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.

A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.

Functional analysis of a G protein-coupled receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.

The myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis (Mollusca) has been the only previously identified myokinin receptor. We had cloned a G protein-coupled receptor (AF228521) from the tick Boophilus microplus (Arthropoda: Acari), 40% identical to the lymnokinin receptor, that we have now expressed in CHO-K1 cells. Myokinins at nanomolar concentrations induced intracellular calcium release, as measured by fluorescent cytometry and the receptor coupled to a pertussis toxin-insensitive G protein. Absence of extracellular calcium did not inhibit the fluorescence response, indicating that intracellular stores were sufficient for the initial response. Control cells only transfected with vector did not respond. We conclude that the tick receptor is the first myokinin receptor to be cloned from an arthropod.

Mares with delayed uterine clearance have an intrinsic defect in myometrial function.

Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F(2alpha) (PGF(2alpha)) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca(2+) regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca(2+) transients of myometrial cells loaded with a Ca(2+)-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca(2+) transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca(2+) concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF(2alpha), suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca(2+) release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.

Evaluation of methods for predicting the toxicity of polycyclic aromatic hydrocarbon mixtures.

Risk assessments of polycyclic aromatic hydrocarbon mixtures are hindered by a lack of reliable information on the potency of both mixtures and their individual components. This paper examines methods for approximating the toxicity of polycyclic aromatic hydrocarbon (PAH) mixtures. PAHs were isolated from a coal tar and then separated by ring number using HPLC. Five fractions (A-E) were generated, each possessing a unique composition and expected potency. The toxicity of each fraction was measured in the Salmonella/mutagenicity assay and the Chick Embryo Screening Test (CHEST). Their abilities to induce ethoxyresorufin-O-deethylase and to inhibit gap junction intercellular communication in rat liver Clone 9 cells were also measured. In the Salmonella/mutagenicity assay, fractions were predicted to have potencies in the order C > D > E > B > A. Toxic equivalency factors (TEFs) for fractions A-E were in the order E > or = D > C > B > A. TEF values were 20,652, 20,929, 441, 306, and 74.1 micrograms of BaP equiv/g, respectively. A lack of agreement between assay-predicted potencies and chemical analysis-predicted potencies was observed with other assays and other methods of calculation. The results demonstrate the limitations of using a single method to predict the toxicity of a complex PAH mixture.

Transcriptional activation of deoxyribonucleic acid polymerase alpha gene expression in MCF-7 cells by 17 beta-estradiol.

Treatment of MCF-7 human breast cancer cells with 17beta-estradiol (E(2)) results in increased DNA synthesis and cell proliferation and enhanced enzyme activities associated with purine/pyrimidine biosynthesis. The mechanism of enhanced DNA polymerase alpha activity was investigated by analysis of the promoter region of this gene. E(2) induced luciferase (reporter gene) activity in MCF-7 cells transfected with pDNAP1, pDNAP2, and pDNAP3 containing -1515 to +45, -248 to +45 and -116 to +45 inserts from the DNA polymerase alpha gene promoter, whereas no induction was observed with pDNAP4 (-65 to +45 insert). The induction response was dependent on cotransfection with estrogen receptor alpha (ER(alpha)), and transactivation was also observed with a mutant ER(alpha) that did not express the DNA-binding domain. Subsequent functional, DNA binding, and DNA footprinting studies showed that a GC-rich region at -106 to -100 was required for E(2)-mediated transactivation, and Sp1 protein, but not ER(alpha), bound this sequence. Transcriptional activation of DNA polymerase alpha by E(2) is associated with ER(alpha)/Sp1 action at a proximal GC-rich promoter sequence, and this gene is among a growing list of E(2)-responsive genes that are induced via ER(alpha)/Sp1 protein interactions that do not require direct binding of the hormone receptor to DNA.

Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters hippocampal astroglia-neuronal gap junctional communication.

Halogenated aromatic hydrocarbons (HAHs) such as dibenzo-p-dioxins are known to alter cognitive function. However, the cellular basis of this disruption is not well understood. One possible deleterious effect of exposure to HAHs could be on gap junctional intercellular communication (GJIC) between neurons and astroglia in the brain. As such, this study examined the effects of the highly toxic prototypic HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on GJIC in rat hippocampal primary cell culture. Initial measurements of fluorescence recovery after photobleaching (gap-FRAP) showed dye transfer between astroglia and neurons. N-octanol, a lipophilic alcohol known to uncouple cells by decreasing the open probability of gap junctional channels blocked astroglial-neuronal (A-N) communication as well as astroglial-astroglial (A-A) communication. TCDD initially downregulated GJIC between neurons and astroglia of treatment, but had no effect on astroglial cell pairs. These results indicate the presence of GJIC between neurons and astroglia in culture and demonstrate different sensitivities of gap junction responses to TCDD in homologous and heterologous cell pairs. The finding that 2,3,7,8-TCDD disrupts GJIC through A-N but not A-A channels may have important implications for impaired brain function resulting from developmental exposure to TCDD.

Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells.

There is great interest in utilizing butyrate as a chemopreventive agent for colon tumorigenesis because of its ability to promote apoptosis in colon tumor cell lines. Because CD95 (APO-1/Fas) transduces signals resulting in apoptosis, we tested the hypothesis that butyrate-dependent colonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas and its ligand in young adult mouse colon (YAMC) cells. To delineate the proapoptotic effect of butyrate and to avoid the confounding effects of detachment from the extracellular matrix, adherent cell apoptosis was monitored as loss of plasma membrane asymmetry and dissipation of mitochondrial membrane potential (DeltaPsi(mt)) by laser cytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspase inhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction of apoptosis. Treatment with Fas agonistic antibody (clone Jo-2) significantly induced cell death, indicating that Fas in colonocytes is functional. In addition, butyrate promoted apoptosis by inducing loss of DeltaPsi(mt) and phospholipid asymmetry of the plasma membrane after 12 and 24 h of exposure, respectively, before cell detachment. Therefore, Fas receptor-dependent signal transduction is involved in butyrate induction of apoptosis in colonocytes.

Immunosuppressant inhibition of P-glycoprotein function is independent of drug-induced suppression of peptide-prolyl isomerase and calcineurin activity.

PURPOSE: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P-glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptideprolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. METHODS: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P-glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. RESULTS: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF-7/Adr cells with CaN and FKBP12 antagonists. CONCLUSIONS: These results demonstrate that the inhibitory effects of immunosuppressive agents on P-glycoprotein function are independent of CaN or FKBP12 PPI activity.

Oxytocin-induced Ca2+ responses in human myometrial cells.

Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of

A glyconutritional mixture (Ambrotose®) provides some amelioration to mice with coxsackievirus-induced pancreatitis.

Challenge of adolescent male CD-1 mice with a coxsackievirus B3 (CVB3) strain (CVB3m) induces mild to severe destruction of pancreatic acinar cells, but causes no deaths and does not induce hyperglycemia. A weekly parenteral (intraperitoneal) administration of a glyconutritional mixture (Ambrotose® to virus-challenged mice was assessed to determine if there were any benefits to recovery over an eight month period. Virus-challenged mice showed a significant weight loss over the initial five weeks of the experiment, but injection of Ambrotose® to similar virus-challenged mice restored the total body weight to levels found in normal mice. Normal mice given Ambrotose® exhibited a small weight gain. Mice given Ambrotose® showed reduced severity of pancreatitis, as evidenced by significant reductions in percentages of pancreatic acinar cells destroyed and proportion of sections of pancreata with destroyed acinar cells, compared to virus control-mice not injected with Ambrotose®. Statistical analyses of the extent of acinar cell pathology in all virus-challenged mice showed that Ambrotose® contributed significantly to recovery of the acinar cell population in virus-inoculated mice. Anti-viral antibody titers were not affected by Ambrotose® injections. One potential mechanism to explain the benefits derived from Ambrotose® injections came from studies of antioxidant levels of glutathione in splenic macrophages/monocytes. Whereas CVB3 challenge of mice reduced glutathione levels in the latter cells, Ambrotose® injections to virus-challenged mice restored glutathione levels to those found in normal mice. In summary, most but not all mice derived benefits from Ambrotose® injections, i.e. a reduction in pathology in the pancreas and restored levels of the antioxidant glutathione in macrophages/monocytes. Higher doses of Ambrotose® could provide greater benefits for more mice, a study for the future.

Protection from glutathione depletion by a glyconutritional mixture of saccharides.

A complex glyconutritional (GN) mixture of mono-, di-and polysaccharides was investigated to assess its capacity to protect two different types of rodent cells, rat hepatocytes and mouse splenocytes, from depletion of glutathione by a sulfhydryl-reactive mycotoxin, patulin, or by coxsackievirus B3 (CVB3) infection, respectively. Rat hepatocytes were treated with the GN mixture in vitro or received carrier medium only prior to treatment with patulin. When treated with the GN mixture prior to patulin exposure hepatocytes demonstrated protection against depletion of intracellular reduced glutathione (GSH). Cells treated with the GN for up to 15 hours prior to patulin exposure showed no increase in protection of GSH above that demonstrated by cells treated for 3 hours. Mice were infected with CVB3 and one treatment group was injected intraperitoneally with the GN once a week. Animals were splenectomized each month over a ten month treatment for analysis of spleen monocytic cells. Splenocytes from mice treated with the GN mixture did not show the virally-associated depletion of intracellular GSH or damage to pancreatic acini observed in CVB3 inoculated but non-GN-treated mice. Animals from which spleen cells were taken for analysis showed no decrease in anti-CVB3 antibodies and no decrease in viral titers to accompany or explain the normal levels of intracellular GSH. These data strongly suggest that a complex mixture of exogenous saccharides exerts a protective effect on liver cells in vitro in that the cells are protected from chemically initiated depletion of intracellular GSH, and on spleen cells in vivo in that the cells are protected against a CVB3-initiated decrease in intracellular GSH and increase in pancreatic acini damage.

Attenuation of gossypol cytotoxicity by cyclic AMP in a rat liver cell line.

The effect of pretreatment of a rat liver cell line (Clone 9) with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), an agent previously shown to increase gap junctional intercellular communication (GJIC), was used to determine if increased GJIC would attenuate the cytotoxic effects of gossypol acetic acid (GAA). Pretreatment with 8-Br-cAMP increased the inhibitory concentration 50% (IC50) for gossypol from 4.1 to 6.1 microM in Clone 9 cells. GJIC was suppressed by about 57% within 5 min when Clone 9 cells were treated with 1.0 microM GAA alone, whereas cells treated with 3.0 or 10 microM GAA were completely uncoupled within the same time frame. In contrast, GJIC was maintained near control levels in Clone 9 cells preincubated with 1.0 mM 8-Br-cAMP for 10 min prior to 1.0 microM GAA exposure. 8-Br-cAMP partially restored GJIC in cells treated with 3.0 microM GAA but was unable to protect cells exposed to 10 microM GAA. The effects of GAA and 8-Br-cAMP pretreatment on connexin43 (Cx43) protein expression was analyzed with Western blots. GAA treatment at concentrations of 1 and 3 microM caused a time- and dose-dependent increase in phosphorylation of Cx43 over a 20-min period, whereas 10 microM GAA caused a time-dependent degradation of Cx43 over the same interval. Pretreatment of cells for 10 min with 8-Br-cAMP completely reversed the effect of 1 microM GAA and partially blocked the effect of 3 microM GAA on Cx43 phosphorylation and suppressed Cx43 degradation at 10 microM. Additional fluorescence endpoints associated with cellular homeostasis mechanisms were also monitored to evaluate cytotoxicity and potential protective effects of 8-Br-cAMP pretreatment in cells exposed to GAA, including generation of reactive oxygen species (ROS), cytoplasmic acidification, glutathione (GSH) content, intracellular calcium levels ([Ca2+]i), and mitochondrial and plasma membrane potential. The adverse effects of GAA on the production of ROS, cytoplasmic acidification, GSH content, and [Ca2+]i were also attenuated. The primary protective effect of 8-Br-cAMP was observed at or below the IC50 of GAA, with greatest protective effects detected on early endpoints affected by GAA exposure. These studies suggest that the protective effect of 8-Br-cAMP in GAA-treated cells results from enhanced GJIC, which facilitates cellular homeostasis by providing cell-cell diffusion of essential metabolites, ions, and regulatory and informational molecules.

Endometrial connexin expression in the mare and pig: evidence for the suppression of cell-cell communication in uterine luminal epithelium.

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction-associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction-mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness.

Doxorubicin induced expression of P-glycoprotein in a canine osteosarcoma cell line.

Canine and human osteosarcoma are very similar with respect to clinical presentation, radiological and histopathological features, metastatic rate and pattern and response to therapy. For these reasons, canine osteosarcoma is a useful intermediate model for the disease in humans. Overexpression of P-glycoprotein, the product of the MDR1 gene, is the most important predictor of an adverse clinical course in human patients with osteosarcoma. Exposure of canine osteosarcoma cells to doxorubicin resulted in overexpression of MDR1 mRNA and P-glycoprotein. Furthermore, these cells failed to accumulate doxorubicin intracellularly and were less sensitive to vincristine-induced cytotoxicity as compared to parental cells.

Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays.

Comparative kinetic analyses of the mechanisms of toxicity of the alkylphenol eugenol and its putative toxic metabolite (quinone methide, EQM) were carried out in cultured rat liver cells (Clone 9, ATCC) using a variety of vital fluorescence bioassays with a Meridian Ultima laser cytometer. Parameters monitored included intracellular GSH and calcium levels ([Ca2+]i), mitochondrial and plasma membrane potentials (MMP and PMP), intracellular pH, reactive oxygen species (ROS) generation, and gap junction-mediated intercellular communication (GJIC). Cells were exposed to various concentrations of test compounds (1 to 1000 microM) and all parameters monitored directly after addition at 15 s intervals for at least 10 min. Eugenol depleted intracellular GSH, inhibited GJIC and generation of ROS, and had a modest effect on MMP at concentrations of 10 to 100 microM. At high concentrations (1000 microM), eugenol also affected [Ca2+]i, PMP, and pH. Effects of EQM were seen at lower concentrations (1 to 10 microM). The earliest and most potent effects of either eugenol or EQM were seen on GSH levels and GJIC. Coadministration of glutathione ethyl ester enhanced intracellular GSH levels by almost 100% and completely protected cells from cell death caused by eugenol and EQM. These results suggest that eugenol mediates its hepatotoxic effects primarily through depletion of cytoprotective thiols and interference in thiol-dependent processes such as GJIC. Furthermore, our results support the hypothesis that the toxic effects of eugenol are mediated through its quinone methide metabolite.

Analysis of Pb2+ entry into cultured astroglia.

Astroglia serve as a presumptive lead (Pb) sink in the brain; therefore, this study examined Pb entry into cultured rat astroglia utilizing the Ca2+ fluorophore indo-1 as a tool for detecting Pb2+ entry during acute exposure. The interactions of Pb2+ with indo-1 were analyzed by fluorescence spectrophotometry in a cell-free system. The emission spectrum of Pb2+/indo-1 was substantially different from that of Ca2+/indo-1 due to suppression of indo-1 fluorescence emission intensity. Next, we established the presence of L-type Ca2+ channels in astroglial cultures and demonstrated that Pb accumulation is enhanced under serum-free conditions and by the application of Bay-K 8644. Because acute exposure is of less toxicologic relevance than repeated low-level exposure, we then examined Pb uptake in cultures treated for up to 1 week with Pb. AAS revealed that Pb accumulation was accompanied by an increase in total cellular [Ca]. In addition, differences in basal indo-1 fluorescence levels and differences in responsiveness to ionomycin were observed. Ionomycin induced an increase in the fluorescence ratio in untreated cells but cells treated for 1 day with Pb showed no response to ionomycin. However, cells treated for 3 and 7 days showed a partial response to ionomycin. TPEN was used to evaluate the interactions of Pb2+ with indo-1 and only cells treated for 7 days showed a response to TPEN. Thus, the present study characterizes Pb2+ entry into astroglia via L-type Ca2+ channels and presents the possibility of using indo-1 for analysis of Pb2+ uptake and the subsequent neurotoxic events in astroglia.

Studies on the relationship between estrogen receptor content, glutathione S-transferase pi expression, and induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and drug resistance in human breast cancer cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces both phase I and phase II drug-metabolizing enzymes in rodent liver and hepatoma cell lines and this induction is mediated by the aryl hydrocarbon (Ah) receptor. Induction of CYP1A1 by TCDD in human breast cancer cells has been reported and results of several studies suggest that the estrogen receptor (ER) may be required for Ah responsiveness. This study investigates the induction of GST pi by TCDD in human breast cancer cells and the role of the ER in mediating this response. TCDD did not induce chloramphenicol acetyl transferase (CAT) activity in ER positive (ER+) MCF-7 and ER- MDA-MB-468 and MDA-MB-231 human breast cancer cell lines transiently transfected with GST pi (human) or GSTP (rat) promoter-reporter constructs containing the -291/+36 and -2.9/+59 region, respectively, of the GST pi and GSTP gene promoters. Furthermore, TCDD did not induce GST pi or GSTP in MDA-MB-468 and MDA-MB-231 human breast cancer cells stably transfected with the ER. RT-PCR confirmed that GST pi mRNA levels were low in ER+ MCF-7 cells and high in ER- MDA-MB-468 and MDA-MB-231 cells; however, in MDA-MB-468 and MDA-MB-231 cells stably transfected with the ER GST pi mRNA levels remained elevated and were not inducible. MDA-MB-468 and MDA-MB-231 cells stably transfected with the ER exhibited increased GST activity and decreased GSH content compared to wild-type cells; however, in MDA-MB-468 cells stably transfected with ER, the susceptibility to doxorubicin, ellipticine, chlorambucil, malphalan, or cisplatin was similar to that observed in wild-type cells. Adriamycin accumulation was similar in wild-type and ER stably transfected cells and verapamil did not affect this response, suggesting that ER expression did not influence p-glycoprotein activity. Taken together these data suggest that not all GST isoforms are responsive to TCDD and stable transfection of ER- cells with ER is not sufficient to restore the ER+ phenotype in some breast cancer cell lines.