Disruption of oogenesis and molting by methoprene and glyphosate in Gammarus fossarum: involvement of retinoic acid?

In the last decade, the freshwater amphipod Gammarus fossarum proved to be a promising sentinel species in active biomonitoring programs to assess the effects of environmental contamination on non-target organisms. Given that the highly conserved retinoid (RETs) metabolism supports many biological functions and is perturbed by xenobiotics and used as biomarker for vertebrates, we explored the RETs functions in the crustacean model Gammarus fossarum. More specifically, we studied the implication of all -trans retinoic acid (atRA) in the reproduction (embryo, oocyte, and juvenile production) and development (success and delay of molting) by exposing G. fossarum females to atRA and citral (CIT), a known inhibitor of RA synthesis. In parallel, we exposed gammarids to methoprene (MET) and glyphosate (GLY), two pesticides suspected to interfere with atRA metabolism and signaling and frequently found in water systems. After 14 days of exposure, atRA, CIT, and MET reduced the number of oocytes, whereas only MET caused a reduced number of embryos. After 44 days, MET and GLY showed a tendency to decrease juvenile production. The duration of the molting cycle increased following the exposures to atRA and MET, while the treatment with CIT caused a typical endocrine disruptive inverted U-shaped curve. The exposure to GLY led to increased duration of the molting cycle at the lowest concentrations and lowered molting success at the highest concentration tested. This study highlights for the first time the implication of RA in the oogenesis and molting of G. fossarum and suggests that it may be a potential mediator of MET-induced effects on these processes. This study adds to the comprehension of the reproductive and developmental control in G. fossarum and opens new research avenues to study the effects of xenobiotics on the RET system in this sentinel species. Ultimately, our study will drive the development of RET-based biomarkers for non-target aquatic invertebrates exposed to xenobiotics.

The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate.

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Cadmium affects autophagy in the human intestinal cells Caco-2 through ROS-mediated ERK activation.

Cadmium is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium has the capacity to accumulate high levels of this metal. We have previously shown that Cd induces ERK1/2 activation in differentiated but not proliferative human enterocytic-like Caco-2 cells. As autophagy is a dynamic process that plays a critical role in intestinal mucosa, we aimed the present study 1) to investigate the role of p-ERK1/2 in constitutive autophagy in proliferative Caco-2 cells and 2) to investigate whether Cd-induced activation of ERK1/2 modifies autophagic activity in postconfluent Caco-2 cell monolayers. Western blot analyses of ERK1/2 and autophagic markers (LC3, SQSTM1), and cellular staining with acridine orange showed that ERK1/2 and autophagic activities both decreased with time in culture. GFP-LC3 fluorescence was also associated with proliferative cells and the presence of a constitutive ERK1/2-dependent autophagic flux was demonstrated in proliferative but not in postconfluent cells. In the latter condition, serum and glucose deprivation triggered autophagy via a transient phosphorylation of ERK1/2, whereas Cd-modified autophagy via a ROS-dependent sustained activation of ERK1/2. Basal autophagy flux in proliferative cells and Cd-induced increases in autophagic markers in postconfluent cells both involved p-ERK1/2. Whether Cd blocks autophagic flux in older cell cultures remains to be clarified but our data suggest dual effects. Our results prompt further studies investigating the consequences that Cd-induced ERK1/2 activation and the related effect on autophagy may have on the intestinal cells, which may accumulate and trap high levels of Cd under some nutritional conditions.

Pesticides Inhibit Retinoic Acid Catabolism in PLHC-1 and ZFL Fish Hepatic Cell Lines.

The population of yellow perch (Perca flavescens) in lake Saint-Pierre (QC, Canada) has been dramatically declining since 1995 without any sign of recovery. Previous studies have shown disrupted retinoid (vitamin A) metabolic pathways in these fish, possibly due to the influence of pesticides. Our study aimed to evaluate the impact of some herbicides and neonicotinoids on retinoic acid catabolism in the fish hepatic cell lines PLHC-1 and ZFL. We hypothesized that pesticides accelerate the catabolism of retinoic acid through oxidative stress that exacerbates the oxidation of retinoic acid. Results obtained with talarozole, a specific CYP26A1 inhibitor, and ketoconazole, a generalist inhibitor of cytochrome-P450 enzymes, revealed that CYP26A1 is mainly responsible for retinoic acid catabolism in ZFL but not PLHC-1 cells. The impacts of pesticides on retinoic acid catabolism were evaluated by incubating the cells with all-trans-retinoic acid and two herbicides, atrazine and glyphosate, or three neonicotinoids, clothianidin, imidacloprid, and thiamethoxam. Intracellular thiols and lipid peroxidation were measured following pesticide exposure. The possible causal relation between oxidative stress and the perturbation of retinoic acid catabolism was investigated using the antioxidant N-acetylcysteine. The data revealed that pesticides inhibit retinoic acid catabolism, with the involvement of oxidative stress in the case of atrazine, imidacloprid, and thiamethoxam but not with clothianidin and glyphosate. Pesticides also affected the isomerization of all-trans-retinoic acid over time, leading to an increased proportion of active isomers. These results hint at a possible perturbation of retinoic acid catabolism in fish living in pesticide-contaminated waters, as suggested by several in vivo studies. Such a disruption of retinoid metabolism is worrying, given the numerous physiological pathways driven by retinoids.

Zinc interference with Cd-induced hormetic effect in differentiated Caco-2 cells: Evidence for inhibition downstream ERK activation.

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium represents an effective protective barrier against Cd toxicity, but it is also a target tissue that may accumulate and trap high levels of the ingested metal. Using human enterocytic-like Caco-2 cells, we have previously shown that Cd may induce a concentration and time-dependent increase in 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT)-reducing activity in differentiated cultures with correlation to ERK1/2 activation. The present study shows that (a) Zn prevents the Cd-induced hormesis effect on MTT reduction in a concentration-dependent manner, without inhibiting Cd-induced ERK1/2 activation; (b) Zn also induces similar hormetic stimulation of MTT-reducing activity but without ERK1/2 activation. The effect of both metals was sensitive to inhibitors of translation during protein synthesis. There is evidence for the involvement of reactive oxygen species (ROS) in Cd-induced ERK1/2 activation. In contrast, the Zn effect on the MTT-reducing activity would not be triggered by ROS but it would be sensitive to the redox state of the cell. Steps downstream ERK1/2 activation by Cd does not involve eIF4E which is rather downregulated by Cd. In conclusion, Cd and Zn both can modify translation processes during protein synthesis via different signaling cascades with crosstalk, and cross-inhibition may occur. This phenomenon is observed over a small range of metal concentrations and is characterized by a hormesis-like response. Considering that the hormetic effect on dehydrogenase activity could reflect an adaptive response to the metals whether cross-inhibition is beneficial is an open question.

Foraging in maize field areas: A risky business?

In Quebec, Canada, the cultivation of maize dominates the agricultural territory. This crop requires a sustained supply of fertilizers from different sources: chemical, natural or from residual materials (sludge). These amendments contain metallic trace elements, which may lead to metal-contaminated maize pollen, a possible source of prooxidants for the foraging bees. Our objective was to determine whether maize fields environment influences the oxidation processes and the accumulation of metals in bees. A few days prior to pollen shedding, beehives were installed in maize fields: one organically grown (site A) and three conventionally grown (sites B, C and D). Soil, maize pollen and bees were analyzed for metal content. Every 15days, bees were collected and analyzed for peroxidation of lipids, metallothionein-like proteins (MTLPs), proteins, retinoids and lipophilic antioxidants (carotenoids and α-tocopherol). The compound ß-carotene was the most abundant in bees from all sites, followed by α-carotene, ß-cryptoxanthin, α-cryptoxanthin, zeaxanthin and lutein. Retinaldehyde and retinol varied according to times and sites without demonstrating clear trends. However, significant differences between sites were noted in 13-cis-retinoic acid and two retinoic acid metabolites measured in bees, suggesting alteration in the reduction-oxidation processes. In line with these results, the level of lipid peroxidation was globally higher in sites B, C and D compared with the organic site. Higher concentrations of metals were observed in soil and pollen from the field A, but bees metal contents were equal or less than those measured in bees from other sites. Higher bee MTLP levels were measured in sites B, C and D. For most sampling times, the discriminant analysis revealed that the conditions were distinguished by the oxidation processes in bees. Our data suggest that bees foraging in conventionally grown maize fields are at risk of increased oxidative damages which can alter the fine regulation of retinoids.

Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H2O2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H2O2, NH3, or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H2O2-induced damage that may occur during histamine oxidative deamination.

Mixtures of herbicides and metals affect the redox system of honey bees.

The increasing loss of bee colonies in many countries has prompted a surge of studies on the factors affecting bee health. In North America, main crops such as maize and soybean are cultivated with extensive use of pesticides that may affect non-target organisms such as bees. Also, biosolids, used as a soil amendment, represent additional sources of metals in agroecosystems; however, there is no information about how these metals could affect the bees. In previous studies we investigated the effects of environmentally relevant doses of herbicides and metals, each individually, on caged honey bees. The present study aimed at investigating the effects of mixtures of herbicides (glyphosate and atrazine) and metals (cadmium and iron), as these mixtures represent more realistic exposure conditions. Levels of metal, vitamin E, carotenoids, retinaldehyde, at-retinol, retinoic acid isomers (9-cis RA, 13-cis RA, at-RA) and the metabolites 13-cis-4-oxo-RA and at-4-oxo-RA were measured in bees fed for 10 days with contaminated syrup. Mixtures of herbicides and cadmium that did not affect bee viability, lowered bee α- and ß-carotenoid contents and increased 9-cis-RA as well as 13-cis-4-oxo-RA without modifying the levels of at-retinol. Bee treatment with either glyphosate, a combination of atrazine and cadmium, or mixtures of herbicides promoted lipid peroxidation. Iron was bioconcentrated in bees and led to high levels of lipid peroxidation. Metals also decreased zeaxanthin bee contents. These results show that mixtures of atrazine, glyphosate, cadmium and iron may affect different reactions occurring in the metabolic pathway of vitamin A in the honey bee.

Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera).

Several hypotheses have been proposed to explain the abnormally high mortality rate observed in bee populations in Europe and North America. While studies based on the effects of pesticides are paramount, the metals present in agroecosystems are often overlooked. Sources of metals are linked to the nature of soils and to agricultural practices, namely the use of natural or chemical nutrients as well as residual materials from waste-water treatment sludge. The aim of this study was to investigate the effects of metals on honey bees exposed for 10 days to environmentally realistic concentrations of Al, Pb and Cd (dissolved in syrup). The monitoring of syrup consumption combined with the quantification of metals in bees revealed the following order for metal bioconcentration ratios: Cd > Pb > Al. Alpha-tocopherol, metallothionein-like proteins (MTLPs) and lipid peroxidation were quantified. When bees were exposed to increasing amounts of Cd, a marked augmentation of MTLPs levels was found. Lead (Pb) and Cd caused an increase in α-tocopherol content, while alteration of lipid peroxidation was observed only with Al exposure. These findings raise concerns about the bioavailability and the additional threat posed by metals for pollinators in agricultural areas while providing new insights for potential use of the honey bee as a sentinel species for metal exposure.

Effects of realistic doses of atrazine, metolachlor, and glyphosate on lipid peroxidation and diet-derived antioxidants in caged honey bees (Apis mellifera).

The decline in the population of pollinators is a worrying phenomenon worldwide. In North America, the extensive use of herbicides in maize and soya crops may affect the health of nontarget organisms like the honey bee. In this study, caged honey bees were exposed to realistic doses of atrazine, metolachlor, and glyphosate for 10 days via contaminated syrup. Peroxidation of lipids was evaluated using the thiobarbituric acid reactive substance (TBARS) test, and diet-derived antioxidants-carotenoids, all-trans-retinol (at-ROH) and α-tocopherol-were detected and quantified using reversed-phase HPLC techniques. Significant increases in syrup consumption were observed in honey bees exposed to metolachlor, and a lower TBARS value was recorded for the highest dose. No relationship was observed between the peroxidation of lipids and the levels of antioxidants. However, ß-carotene, which was found to be the most abundant carotenoid, and at-ROH (derived from ß-carotene) both decreased with increasing doses of atrazine and glyphosate. In contrast, metolachlor increased levels of at-ROH without any effects on ß-carotene. These results show that the honey bee carotenoid-retinoid system may be altered by sublethal field-realistic doses of herbicides.

Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441.

Sensitivity to Cd and Zn as well as the capacity to develop tolerance were characterized in human lung cells A549 and H441. In the A549 cells, a 2-fold lower LC(50) was obtained for Cd compared to Zn, whereas H441 cells were similarly sensitive to both metals. H441 cells were twice as resistant to Cd as the A549 cells. Higher HSP70, but not metallothionein (MT) or glutathione (GSH) levels, could contribute to this better resistance. A 1.5- and 2-fold increase in the LC(50) for Cd was obtained in the A549 cells pre-exposed to non-cytotoxic concentrations of Cd (20 µM) or Zn (40 µM) for 24 h. On the other hand, only Zn increased H441 cells' resistance to Cd. Maximum Zn- and Cd-induced tolerances were reached as early as 3 and 12 h, respectively. Increases in MT-IIa and HSP70 messenger RNA levels were higher in A549 cells, but cycloheximide eliminated the induction of tolerance only in the H441 cells. Protein synthesis is a prerequisite for metal-induced tolerance to Cd in the H441 cells but not the A549 cells. Results obtained with L-buthionine sulfoximine revealed that GSH synthesis is not responsible for the acquired tolerance in both cell lines. However, GSH plays a critical role against Cd toxicity, and pro-oxidant conditions sensitized cells to Cd with different impacts on the metal-induced mechanisms of acquired tolerance. GSH and catalase both provide antioxidative protection, but only the stress related to low GSH content, not that resulting from catalase inhibition, may be alleviated with Zn.

Expression of macrophage migration inhibitory factor by osteoblastic cells: protection against cadmium toxicity.

Exposition to cadmium (Cd) has been linked to bone metabolism alterations and occurrence of osteoporosis. Despite its known renal toxicity which indirectly disrupts bone metabolism through impairment of vitamin D synthesis, increasing evidence argues for the direct action of Cd on bone-forming osteoblasts. Indeed, accumulation of Cd in osteoblasts and metal-induced cell death has been documented but little is known about the intracellular mechanisms of protection against this stress. In this work, we investigated the protection afforded by thiol-containing proteins against Cd cytotoxicity in MC3T3 osteoblastic cells. Viability of MC3T3 cells was reduced by Cd in a concentration-dependent manner with a LC(50) of 7.6±1.1µM. Depletion of glutathione by l-buthionine sulphoximine (BSO) increased cell sensitivity to Cd cytotoxicity, suggesting the involvement of thiol-containing peptides as a mechanism of protection. Accordingly, Cd was shown to promote progressive depletion of reduced thiol content and to stimulate the production of reactive oxygen species (ROS). Interestingly, low non cytotoxic concentrations of Cd increased the gene expression of macrophage migration inhibitory factor (MIF), also a thiol-containing protein. Inhibition of the transcription factor NFκB prevented Cd-dependent upregulation of MIF expression and consequently, increased Cd cytotoxicity in osteoblasts. Moreover, MIF deficient mouse osteoblasts were more sensitive to Cd cytotoxicity than the corresponding control cells. By gel-filtration chromatography, we demonstrated that MIF acts as a thiol-containing protein and thereby promotes Cd complexation. In accordance with its binding ability, addition of recombinant MIF to the culture medium reduced Cd cytotoxicity. Overall, upregulation of MIF expression by Cd may protect against the cytotoxicity of this metal in the osteoblasts.

Fe- and Zn-induced inhibition of Cd uptake in human lung cell lines: speciation studies with H441 and A549 cells.

Cadmium transport was studied in human lung cell lines A549 and H441 as a function of inorganic metal speciation. A 2-fold higher equilibrium accumulation was obtained in the A549 cells, but a specific system of transport of high affinity and low capacity was characterized in both these cells. Exposure conditions optimizing [Cd2+] increased Cd uptake, but CdCln2-n species are also taken up. Studies on Cd-Ca reciprocal inhibition do not support a role for Ca pathways in Cd uptake. Nramp2 and Zip8 mRNAs were detected in both cell lines. Fe inhibited Cd uptake in the nitrate medium with an apparent at pH 5.5. NRAMP2 may contribute to Cd uptake but a major role is precluded since the overall process of accumulation was inhibited by acidic conditions. Zn and Mn inhibited Cd uptake with the following observations: (1) Similar apparent Ki values for Zn-induced inhibition in the chloride and nitrate media. (2) Whatever the medium, a higher Mn-induced inhibition at pH 5.5 compared to pH 7.4, with much lower Ki values under acidic conditions. (3) Elimination of the stimulatory effect of HCO3- by Zn but not by Mn. Zn may inhibit a ZIP8-mediated Cd uptake, whereas the Mn-sensitive component of uptake would be related to other transport processes.

Characterization of basolateral-to-apical transepithelial transport of cadmium in intestinal TC7 cell monolayers.

Cadmium (Cd) is a toxic metal with an extremely long half-life in humans. The intestinal absorption of Cd has been extensively studied but the role the intestinal epithelium may play in metal excretion has never been considered. The basolateral (BL)-to-apical (AP) transepithelial transport of Cd was characterized in TC7 human intestinal cells. Both AP and BL uptakes varied with days in culture, and BL uptake was twofold higher compared to AP in differentiated cultures. A 50% increase in the BL uptake of 0.5 µM (109)Cd was observed at pH 8.5 in a chloride but not nitrate medium, suggesting the involvement of a pH-sensitive mechanism of transport for chloro-complexes. Fe and Zn inhibited the BL uptake of Cd whereas complexation by albumin had no effect, but the stimulatory effect of pH 8.5 was lost in the presence of albumin. The BL uptake of [(3)H]-MPP(+) and (109)Cd were both inhibited by decynium22 without reciprocal inhibition. MRP2 and MDR1 mRNA levels increased as a function of days in culture. A 25 and 20% decrease in the cellular AP efflux of Cd was observed in the presence of verapamil and probenecid, respectively. In cells treated with BSO, which lowered by 26% the total cellular thiol content, the inhibitory effect of verapamil increased, whereas that of probenecid decreased. These results reveal the existence of a decynium22-sensitive mechanism of transport for Cd at the BL membrane, and suggest the involvement of MDR1 and MRP2 in cellular Cd efflux at the AP membrane. It is conceivable that the intestinal epithelium may contribute to Cd blood excretion.

Involvement of transient receptor potential melastatin-related 7 (TRPM7) channels in cadmium uptake and cytotoxicity in MC3T3-E1 osteoblasts.

Exposure to cadmium (Cd) disrupts bone metabolism, causing osteoporosis. Impaired vitamin D metabolism was initially proposed as the underlying mechanism, yet recent studies argue for the direct effect of Cd on bone cells. This study aimed at characterizing (109)Cd uptake and cytotoxicity in MC3T3-E1 osteoblasts. Time-dependent accumulation of (109)Cd was observed with a 50% lethal concentration (LC(50)) of 9.6 ± 1.2 µM at 24-h. Reducing extracellular calcium (Ca) or magnesium (Mg) increased Cd cytotoxicity. The presence of Ca, Mg, zinc or gadolinium decreased (109)Cd uptake suggesting the involvement of non-selective cationic channels. The Mg-sensitive part of (109)Cd uptake increased at acidic pH, a condition known to stimulate TRPM7 channel activity. Stimulating TRPM7 channel activity by cellular Mg starvation enhanced (109)Cd uptake. Silencing TRPM7 channel expression abolished the Mg-sensitive and the Mg starvation-induced uptake indicating that TRPM7 is involved in Cd transport in osteoblasts.

Cadmium-induced hormetic effect in differentiated Caco-2 cells: ERK and p38 activation without cell proliferation stimulation.

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium may in part protect against Cd toxicity but is also a target tissue. Using human enterocytic-like Caco-2 cells, we have previously shown differences in sensitivity to Cd according to the differentiation status. The present study focuses on Cd effects on differentiated cells. Concentration and time-dependent increases in MTT (3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) activity were observed in post-confluent cultures exclusively, with a twofold maximal stimulation in 21-day-old cells exposed to 10 microM Cd for 24 h. No concomitant increase in [methyl-(3)H] thymidine incorporation was noted and Cd did not modify cell distribution in the cell-cycle phases. However, Cd-induced increase in MTT activity was inhibited by cycloheximine as well as by inhibitors of ERK1/2 and p38, but not by that of JNK. Consistently, Cd increased the levels of ERK1/2 and p38 phosphorylation. Inhibition of Ras-GTP or PI3K enhanced the stimulatory effect of Cd, whereas mTOR inhibition had no effect. Inhibition of G protein-phospholipase and PKC decreased MTT stimulation. These results show a hormesis-like stimulation of Cd on MTT activity in differentiated intestinal cells exclusively. This effect is not related to cell proliferation but more likely to increased protein synthesis which involves ERK1/2 and p38 cascades and possibly PLC-beta signaling pathways. Because growth-related differentiation of intestinal cells is linked to the selective and sequential activation of MAPKs, the impacts that these Cd-induced perturbations in signaling pathways may have on intestinal functions clearly deserve to be investigated.

Resistance to cadmium as a function of Caco-2 cell differentiation: role of reactive oxygen species in cadmium- but not zinc-induced adaptation mechanisms.

Cadmium (Cd) is a highly toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium is the first biological barrier crossed by Cd and is also an important target tissue. In the present study, the human intestinal Caco-2 cell line was used to evaluate the impact of a low level of exposure on both undifferentiated and differentiated intestinal cells. As revealed by the LC(50) values estimated with the 3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, mature Caco-2 cells were more resistant to Cd. However, following a 24-h exposure to non-cytotoxic levels of Cd (10 microM) or zinc (Zn, 100 microM), threefold increases were obtained in the LC(50) values of 7-day-old cells, whereas increased resistance in 21-day-old cells was observed exclusively with Zn. Induction of MT-IIa and HSP70 mRNAs was higher in undifferentiated cells and an increase in cellular glutathione (GSH) content was observed exclusively in these cell cultures. However, the results obtained with cycloheximide used for inhibiting protein synthesis and with L-buthionine sulfoximine (BSO), which inhibits GSH synthesis, revealed that protein synthesis is not a prerequisite to the development of resistance. The presence of 100 mM 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, prevented Cd-induced but not Zn-induced resistance, as well as sensitized cells to Cd toxicity. These results show for the first time differences in constitutive and acquired resistance to Cd as a function of enterocytic differentiation status and suggest the involvement of different mechanisms for Cd- and Zn-induced adaptation in the intestinal cells. Redox signals may trigger Cd-induced adaptation mechanisms but pro-oxidant conditions would eliminate proliferative intestinal cells capability to develop resistance. This would be critical for Cd- but not Zn-induced mechanisms of resistance since Cd but not Zn may cause oxidative stress.

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells.

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of (109)Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd(2+) species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.

Reciprocal inhibition of Cd(2+) and Ca(2+) uptake in human intestinal crypt cells for voltage-independent Zn-activated pathways.

Cadmium-Ca-Zn interactions for uptake have been studied in human intestinal crypt cells HIEC. Our results failed to demonstrate any significant cross-inhibition between Cd and Ca uptake under single metal exposure conditions. However, they revealed a strong reciprocal inhibition for a Zn-stimulated mechanism of transport. Optimal stimulation was observed under exposure conditions that favor an inward-directed Zn gradient, suggesting activation by extracellular rather than intracellular Zn. The effect of Zn on the uptake of Ca was concentration-dependent, and zinc-induced stimulation of Cd uptake resulted in a 3- and 5.8-fold increase in the K(m) and V(max) values, respectively. Neither basal nor Zn-stimulated Ca uptakes were sensitive to membrane depolarization. However, the stimulated component of uptake was inhibited by the trivalent cations Gd(3+), and La(3+) and to a lesser extent by Mg(2+) and Ba(2+). RT-PCR analysis as well as uptake measurement performed with extracellular ATP and/or suramin do not support the involvement of purinergic P2X receptor channels. Uptake and fluorescence data led to the conclusion that Zn is unlikely to trigger Ca influx in response to Ca release from thapsigargin-sensitive intracellular pools. Our data show that Zn may potentiate Cd accumulation in intestinal crypt cells through mechanism that still needs to be clarified.

Effects of Cu on plasma cortisol and cortisol secretion by adrenocortical cells of rainbow trout (Oncorhynchus mykiss).

Fish are exposed to multiple stressors, often acting concurrently, in their environment. To evaluate the potential of Cu to act as a chemical stressor, rainbow trout (Oncorhynchus mykiss) were exposed to Cu (30 or 80 microg/l) for 30 days in the laboratory and they were subjected to a physical stressor (1 min air exposure) before sampling. Physiological stress indicators in the whole fish as well as cortisol secretion by adrenocortical cells in vitro were measured. Fish exposed to Cu had a lower condition factor, hepatosomatic index, plasma glucose, hepatic glycogen and gill Na(+)/K(+)-ATPase activity compared to controls. Exposure to Cu did not have an effect on basal plasma cortisol (fish sampled without air exposure stress) however, the air exposure-induced increase in plasma cortisol was lower in fish exposed to Cu. Cortisol secretion stimulated by ACTH in vitro was greater in adrenocortical cells isolated from fish exposed to Cu in vivo but in vitro exposure to Cu consistently impaired cortisol secretion. Our results indicate that Cu at high concentrations disrupts cortisol secretion through a direct toxic effect on adrenocortical cells while low concentrations resulting from a 30-day exposure to environmentally relevant Cu concentrations enhances cortisol secretion in response to ACTH in vitro.