Characterization of Butyrate Uptake by Nontransformed Intestinal Epithelial Cell Lines

Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon. Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells. Uptake of ¹⁴C-BT by IEC-6 cells was (1) time- and concentration-dependent; (2) pH-dependent; (3) Na⁺-, Cl⁻- and energy-dependent; (4) inhibited by BT structural analogues; (5) sensitive to monocarboxylate transporter 1 (MCT1) inhibitors; and (6) insensitive to DIDS and amiloride. IEC-6 cells express both MCT1 and Na⁺-coupled monocarboxylate transporter 1 (SMCT1) mRNA. We conclude that ¹⁴C-BT uptake by IEC-6 cells mainly involves MCT1, with a small contribution of SMCT1. Acute exposure to ethanol, acetaldehyde, indomethacin, resveratrol and quercetin reduced ¹⁴C-BT uptake. Chronic exposure to resveratrol and quercetin reduced ¹⁴C-BT uptake but had no effect on either MCT1 or SMCT1 mRNA levels. Uptake of ¹⁴C-BT by FHC cells was time- and concentration-dependent but pH-, Na⁺-, Cl⁻- and energy-independent and insensitive to BT structural analogues and MCT1 inhibitors. Although MCT1 (but not SMCT1) mRNA expression was found in FHC cells, the characteristics of ¹⁴C-BT uptake by FHC cells did not support either MCT1 or SMCT1 involvement. In conclusion, uptake characteristics of ¹⁴C-BT differ between IEC-6 and FHC cells. IEC-6 cells demonstrate MCT1- and SMCT1-mediated transport, while FHC cells do not.     

Heavy-metal (Zn,Cd) tolerance in selected clones of duck weed (Lemna minor)

Cryo-microprobe analysis of quench-frozen fronds of a Zn-tolerant clone of Lemna minor exposed to a high level of Zn (300 μM) showed the presence of cellular deposits consisting of Zn, Mg, K and P or Zn, K and P (Zn phytate). The same Zn-tolerant clone of Lemna minor, when exposed to a high level of Cd (30 μM), showed the presence of globular deposits consisting of Cd, K and P in mature fronds, but the immature cells of the enclosed daughter fronds contained relatively large deposits with Cd and S as the main components (Cd-phytochelatin?). Selection for Zn tolerance in a population of Lemna minor was easily achieved but selection for Cd tolerance has so far not been successful. The Zn-tolerant clone also tolerates high levels of phosphate.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57±17 μM and a transport capacity of 1867±296 nmol mg membrane protein⁻¹ min⁻¹. At higher zinc levels (>500 μM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q₁₀ values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)⁺) at all Zn(II) concentrations examined.     

Lead uptake,toxicity and accumulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> plants

The effects of lead were investigated in bean plants (Phaseolus vulgaris L. cv. Zlota Saxa) grown hydroponically in nutrient solution and exposed to Pb(NO₃)₂ (0.1, 0.5, 1 mM) with or without equimolar concentrations of chelator ethylenediaminetetraacetic acid (EDTA). The roots treated only with Pb(NO₃)₂ accumulated up to 25 g(Pb) kg⁻¹(d.m.), during 4-d exposure. However, in bean plants exposed to 0.5 mM Pb + 0.5 mM EDTA or 1 mM Pb + 1 mM EDTA 2.5 times less Pb was determined. In bean plants treated only with Pb, less than 6 % of total lead accumulated was transported to the aboveground parts, while in the case of plants grown with Pb + EDTA, around 50 % of total Pb was transported to the shoots.     

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.     

Characterization of Arsenate Uptake by Cultured Primary Rat Astrocytes

Arsenate is known to be accumulated by cultured astrocytes and to stimulate astrocytic glutathione export, but the arsenate uptake into astrocytes has not been characterized so far. To address this topic, we have exposed primary rat astrocyte cultures to arsenate and determined the cellular arsenic content by atomic absorption spectroscopy. Viable astrocytes accumulated arsenate in a time- and concentration-dependent manner. Their cellular arsenic content increased almost proportional with time for up to 60 min after application of arsenate. Analysis of the concentration-dependent increase in the specific arsenic content of the cells after 30 min of arsenate exposure revealed that cultured astrocytes take up arsenate with saturable kinetics by a transport process that has apparent K_M- and V_max-values of 1.7 ± 0.2 mM and 28 ± 4 nmol/(mg protein × 30 min), respectively. Arsenate uptake in viable astrocytes was strongly inhibited by the presence of phosphate or by lowering the incubation temperature to 4 °C and was completely abolished in a sodium ion-free medium. These results strongly suggest that the saturable temperature-dependent arsenate uptake into astrocytes is mediated by a sodium-dependent phosphate cotransporter.     

Molecular characterization of ten zinc (Zn) transporter genes and their regulation to Zn metabolism in freshwater teleost yellow catfish Pelteobagrus fulvidraco

BackgroundZn is an essential trace element for vertebrates, and Zn uptake and transport is related with the ZIP family of Zn transporters. Meantime, Zn also influenced the expression of ZIP family members.MethodsWe cloned and characterized the full-length cDNA sequences of ten Zn transport-relevant genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14) from yellow catfish Pelteobagrus fulvidraco, investigated their mRNA tissue expression. These ZIP mRNA expression was also assessed in the primary hepatocytes and intestinal epithelial cells of yellow catfish in response to three Zn levels (0, 30 μM and 60 μM, respectively).ResultsAll these genes shared the similar domains with the corresponding members in mammals. The mRNA expression of the ten ZIP genes was detected in nine-tested tissues, but variable among these tissues. Flow cytometry analysis and confocal microscopy observation indicated that intracellular free Zn²⁺ concentration in hepatocytes and intestinal epithelial cells increased with increasing Zn incubation concentration at both 24 h and 48 h. Zn incubation differentially influenced mRNA levels of ZIP transporters in the hepatocytes and intestinal epithelial cells, in a time- and cells-dependent manners. In the hepatocytes, at 24 h, compared to the control, Zn addition down-regulated mRNA levels of ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP11 and ZIP14; however, ZIP10 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, mRNA levels of ZIP1, ZIP6, ZIP7, ZIP9, ZIP10 and ZIP14 declined with increasing Zn incubation concentrations; ZIP3 mRNA levels were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. In the intestinal epithelial cells, at 24 h, Zn addition down-regulated mRNA levels of ZIP1, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14; ZIP3 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, Zn addition up-regulated mRNA levels of ZIP6 and ZIP9, but down-regulated mRNA levels of ZIP8, ZIP10 and ZIP13. ZIP7, ZIP11 and ZIP14 mRNA abundances were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group.ConclusionFor the first time, our study characterized ten ZIP family members in yellow catfish, explored their mRNA tissue expression. Their regulation to Zn addition were also investigated in the hepatocytes and intestinal epithelial cells of yellow catfish. Our study revealed the mechanism of cells exposed to Zn addition and provided novel insights for the regulatory mechanism of Zn homeostasis.     

Compartmentation of Zinc in Roots and Leaves of the Zinc Hyperaccumulator Thlaspi caerulescens J & C Presl

Thlaspi caerulescens is a metallophyte that is able to hyperaccumulate Zn. In the present study the subcellular compartmentation of Zn was investigated in roots and leaves of this species by means of X-ray microanalysis. Leaves accumulated higher average Zn concentrations than roots. In roots of plants exposed to 10 μM Zn, Zn concentrations in the apoplast were similar to those in vacuoles, while in plants treated with 100 μM Zn considerably higher Zn concentrations were detected in vacuoles than in the apoplast. In epidermal and sub-epidermal cells of leaves of plants from both treatments, Zn mainly accumulated in vacuoles and, to a lesser extent, in the apoplast. In vacuoles from plants exposed to 100 μM Zn, high Zn concentrations were associated with variable amounts of P, Ca and K. In leaves, the highest Zn concentrations (13,600 μg g^?1 d.m.) were found in globular crystals present in many vacuoles of epidermal and subepidermal cells. Smaller deposits with a variable Zn concentration between 1,000 and 18,300 μg g^?1 d.m. were observed in the epidermal and subepidermal cells of roots. Both the high Zn/P element ratios found in the crystals and the absence of Mg indicate that, in contrast to other plant species, myo-inositol hexaphosphate (phytate) is not the main storage form for Zn in Thlaspi caerulescens.     

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln⁴²⁰. A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser¹³⁵ was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC. folic acid; folate absorption; methotrexate     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

Interaction of Cd and Zn during uptake and loss in the polychaete Capitella capitata: Whole body and subcellular perspectives

The interaction between Cd and Zn in aquatic organisms is known to be highly variable. The purpose of this study was to use a subcellular compartmentalization approach to examine Cd and Zn interactions in the deposit-feeding polychaete Capitella capitata (sp. I). Laboratory-reared C. capitata were co-exposed to Cd (background or 50 μg Cd l^− 1) and Zn (background or 86 μg Zn l^− 1) with ¹⁰⁹Cd and ⁶⁵Zn as radiotracers for 1 week. After the 1-week uptake period, subsets of worms were allowed to depurate accumulated metals for an additional 1 week. Worms from both phases (uptake and loss) were then subjected to subcellular fractionation to determine the compartmentalization of metals as metal-sensitive fractions [MSF — organelles and heat-denaturable proteins (HDP)] and biologically detoxified metals [BDM — heat-stable proteins (HSP) and metal-rich granules (MRG)]. Uptake and loss of Cd and Zn in C. capitata at the whole body level were similar at bkgd-Cd/bkgd-Zn, with worms depurating the majority of accumulated metal (∼ 75% Cd and ∼ 64% Zn). When exposure of Zn or Cd was increased (bkgd-Cd/86-Zn; bkgd-Zn/50-Cd), uptake of background levels of Cd or Zn, respectively, was suppressed by ∼ 50%. These accumulated metals, however, were retained during the loss phase resulting in ∼ 40-50% greater Cd and Zn whole body tissue burdens than those of bkgd-Cd/bkgd-Zn worms. Beyond exhibiting similar patterns of uptake and loss at the whole body level, Cd and Zn behaved similarly at the subcellular level. Under background levels (bkgd-Cd/bkgd-Zn), after uptake, worms partitioned a majority of Cd (∼ 65%) and Zn (∼ 55%) to the HSP and organelles fractions. The HDP and MRG fractions contained less than ∼ 6% of both metals. Following depuration, at bkgd-Cd/bkgd-Zn, Cd and Zn were lost from all subcellular fractions; loss from HSP was the greatest contributor to whole body loss. When exposed to elevated concentrations of Zn or Cd, the suppression in uptake of bkgd-Cd or bkgd-Zn observed in whole body uptake was largely due to suppressions in the storage of Cd and Zn to HSP. These results suggest that Cd-Zn interactions reduce partitioning of both Cd and Zn to HSP, indicating that metal-binding proteins such as metallothioneins play a key role in these interactions.     

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin.The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties.Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [¹⁴C]choline uptake inhibition (IC₅₀ = 236.1 ± 1.28 μmol/L, and 217.4 ± 1.33 μmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ± 0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC₅₀ value i.e. inhibiting the growth of 50% of the cells (IC₅₀ = 0.72 ± 1.31 μmol/L).Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.     

Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress

In purified system zinc has been shown to have an antioxidant role. Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined. Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150μM as Zinc chlorur ZnCl₂). In comparison, cell controls were grown in standard culture media (6.5μM Zn). The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330±120 ng/mg protein in 150-μM Zn-treated cells versus 331±21 ng/mg protein in control cells). The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased. The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 μmol/g protein in 150μM Zn-treated cells versus 1.59 μmol/g protein in controls). At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities. Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage. However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H₂O₂), an increased toxicity in Zn-supplemented cells was observed. When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium. If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes.     

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts

At 5 μg/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1–20 μM). hydrogen peroxide (500 μM), and methylene blue (50 μM) mimic the effect of insulin in CEF. PCMB-S,¹ a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1–1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80–100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.     

The effect of lead on protein and DNA metabolism of normal and lead-adapted rat kidney cells in culture

Cultures of epithelial rat kidney cells were used to investigate the effect of lead on protein and DNA metabolism. Lead adapted cells were cultured in 5 and 10 μM Pb(NO₃)₂ for several months and subsequently showed the same growth characteristics but a higher resistance to increased lead doses than untreated cells. Both normal and adapted cell types were exposed to lead nitrate in concentrations from 5 to 1000 μM for incubation periods of from 1 to 24 h. ³H-leucine incorporation was increased in 10 μM lead by up to 140% in normal but not in adapted cells, pointing to a resistance forming mechanism. Not only is the overall rate of protein synthesis increased, but separations of cytosolic proteins by SDS-polyacrylamide electrophoresis indicate the novel or increased synthesis of several proteins. A difference could also be observed in the DNA metabolism, where normal cells show a very pronounced increase of up to 900% after 24 h of incubation. This was not observed in adapted cells.     

Calcium Homeostasis in Digitonin-Permeabilized Bovine Chromaffin Cells

The regulation of cytosolic calcium was studied in digitonin-permeabilized chromaffin cells. Accumulation of ⁴⁵Ca²⁺ by permeabilized cells was measured at various Ca²⁺ concentrations in the incubation solutions. In the absence of ATP, there was a small (10–15% of total uptake) but significant increase in accumulation of Ca²⁺ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca²⁺ into carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool—mainly mitochondria—was active when the calcium concentration was > 1 μM and was not saturated at 25 μM. The Ca²⁺ sequestered by the CCCP-insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP-insensitive pool had a high affinity for calcium, with an EC₅₀ of ～1 μM. When the Ca²⁺ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 μM), the presumed endoplasmic reticulum pool was responsible for ～90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5–10 μM), most of the Ca²⁺ (>95%) went into the CCCP-sensitive pool.     

Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent,c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway

Advanced oxidation protein products (AOPPs), a novel protein marker of oxidative damage, have been confirmed to accumulate in patients with inflammatory bowel disease (IBD), as well as those with diabetes and chronic kidney disease. However, the role of AOPPs in the intestinal epithelium remains unclear. This study was designed to investigate whether AOPPs have an effect on intestinal epithelial cell (IEC) death and intestinal injury. Immortalized rat intestinal epithelial (IEC-6) cells and normal Sprague Dawley rats were treated with AOPP-albumin prepared by incubation of rat serum albumin (RSA) with hypochlorous acid. Epithelial cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit activity, reactive oxygen species (ROS) generation, apoptosis-related protein expression, and c-jun N-terminal kinase (JNK) phosphorylation were detected both in vivo and in vitro. In addition, we measured AOPPs deposition and IEC death in 23 subjects with Crohn''s disease (CD). Extracellular AOPP-RSA accumulation induced apoptosis in IEC-6 cultures. The triggering effect of AOPPs was mainly mediated by a redox-dependent pathway, including NADPH oxidase-derived ROS generation, JNK phosphorylation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Chronic AOPP-RSA administration to normal rats resulted in AOPPs deposition in the villous epithelial cells and in inflammatory cells in the lamina propria. These changes were companied with IEC death, inflammatory cellular infiltration, and intestinal injury. Both cell death and intestinal injury were ameliorated by chronic treatment with apocynin. Furthermore, AOPPs deposition was also observed in IECs and inflammatory cells in the lamina propria of patients with CD. The high immunoreactive score of AOPPs showed increased apoptosis. Our results demonstrate that AOPPs trigger IEC death and intestinal tissue injury via a redox-mediated pathway. These data suggest that AOPPs may represent a novel pathogenic factor that contributes to IBD progression. Targeting AOPP-induced cellular mechanisms might emerge as a promising therapeutic option for patients with IBD.