Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Involvement of reactive oxygen stress in cadmium-induced cellular damage in Euglena gracilis

Inorganic cadmium (Cd) causes cellular damage to eukaryotes and to tissues of higher organisms, including DNA strand breaks and intracellular membrane damage, as a result of reactive oxygen stress. We previously reported cadmium chloride (CdCl2)-induced abnormal cell morphologies in the unicellular eukaryote Euglena gracilis Z (a plant cell model) and its achlorophyllous mutant SMZ strain (an animal cell model). The present study was undertaken to examine whether exposure of both strains to CdCl2 would lead to similar cellular responses, especially with regard to reactive oxygen stress loading and cellular damage. The results indicate that CdCl2 exposure can induce morphological alteration, linked to reactive oxygen stress. Both E. gracilis Z and SMZ cells subjected to short-term, high-dose CdCl2 exposure showed long 'comet lengths' in the so-called 'Comet' assay, indicating DNA strand breaks. Similarly, short-term, high-dose CdCl(2)-exposed cells and CdCl(2)-induced morphologically altered cells showed intense fluorescence of dihydrofluorescein (HFLUOR) after incubation with dihydrofluorescein diacetate (HFLUOR-DA). Positive data on the generation and involvement of intracellular reactive oxygen species (ROS) were obtained from long-term, low-dose CdCl(2)-exposed E. gracilis Z and SMZ, by thiobarbituric acid (TBA)-malondialdehyde (MDA) complex analyses.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

Abstract

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Light dependency of resistance to ionizing radiation in Euglena gracilis

The resistance of Euglena gracilis strains Z (wild type) and SM-ZK (chloroplast-deficient mutant) to ionizing radiation was investigated. The colony forming ability of E. gracilis strain Z was higher than that of strain SM-ZK after 60Cogamma-irradiation. For both strains, the resistance of light-grown cells was higher than that of dark-grown cells, suggesting that the light conditions during culture contribute to the radiation resistance of E. gracilis. The comet assay showed that the ability of rejoining DNA double-strand breaks (dsb) was much higher in the light-grown cells. These results suggest that E. gracilis possesses a light-induced repair system to cope with DNA dsb.     

Hydroquinone-induced genotoxicity and oxidative DNA damage in HepG2 cells

Hydroquinone (HQ) is used as an antioxidant in rubber industry and as a developing agent in photography. HQ is also an intermediate in the manufacture of rubber, food antioxidant and monomer inhibitor. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of HQ and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. DNA strand breaks and DNA-protein crosslinks (DPC) were measured by the proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. Using the SCGE assay, a significant dose-dependent increment in DNA migration was detected at concentrations of HQ (6.25-25 microM); but at the higher tested concentrations (50 microM), a reduction in the migration compared to the maximum migration at 25 microM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of HQ (50 microM). A significant increase of the frequency of micronuclei was found in the range from 12.5 to 50 microM in the micronucleus test (MNT). The data suggested that HQ caused DNA strand breaks, DPC and chromosome breaks. To elucidate the oxidative DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were chosen to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25-50 and 6.25-50 microM, respectively. Moreover, HQ significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 12.5 to 50 microM. All these results demonstrate that HQ exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress. GSH, as a main intracellular antioxidant, is responsible for cellular defense against HQ-induced DNA damage.     

Participation of the inositol phospholipid signaling pathway in the increase in cytosolic calcium induced by tributyltin chloride intoxication of chlorophyllous protozoa Euglena gracilis Z and its achlorophyllous mutant SM-ZK

Exposure of tri-n-butyl tin chloride (TBTCl) as a stressor to Euglena gracilis Z causes rapid alteration of cell morphology followed by deflagellation. The present study was undertaken to reveal the mechanism of the cell response at a molecular level. Chlamydomonas reinhardtii, in this study E. gracilis Z and its achlorophyllous mutant SM-ZK, gave similar results when subject to the same stressor. Indeed, similar results were obtained with both strains. Next, assuming that the morphological alteration caused by TBTCl is mediated by the inositide phosphate-lipid signaling pathway, the effects of signal transduction and Ca2+ release reagents (mastoparan as a G-protein activator, neomycin as a phospholipase C inhibitor, verapamil as a Ca2+ channel blocker, and A23187 as a Ca2+ ionophore) on morphology and intracellular Ca2+ levels were examined with or without TBTCl. The data strongly suggest that the morphological alteration is mediated by an increase in Ca2+ linked to the inositol phosphatide pathway. The cellular response to signal transduction inducing reagents was compared between the E. gracilis chlorophyllous Z strain and its achlorophyllous mutant SM-ZK strain. Significant differences were observed between the Z and SM-ZK strains in terms of the stress response and intracellular Ca2+ level.     

Role of oxidative stress and thiol antioxidant enzymes in nickel toxicity and resistance in strains of the green alga Scenedesmus acutus f. alternans.

Treatment with Ni(NO3)2 leads to the formation of reactive oxygen species (ROS) in the green alga Scenedesmus acutus f. alternans, causing lipid peroxidation. This effect was stronger in a Ni-sensitive strain, UTEX72, than in a Ni-resistant strain, B4. In the resistant strain, Ni induced an increased ratio of reduced to oxidized glutathione (GSH:GSSG), whereas it caused a lowered ratio in the sensitive strain. Enzymes involved in the control of ROS were studied in these strains as well as two others that have shown different degrees of nickel resistance. The resistant strain, B4, which grows while containing large amounts of internal Ni, had much higher levels of glutathione reductase and catalase than the other strains. The sensitive strain, UTEX72, had higher levels of glutathione peroxidase, superoxide dismutase, and glucose-6-phosphate dehydrogenase than did strain B4. The resistant strains, Ni-Tol and Cu-Tol, derived from strain UTEX72, which are partly able to exclude Ni, had enzyme profiles that resembled that of UTEX72 more closely than that of B4. Treatment with 10 and 100 microM Ni for 4 or 22 h had complex effects on enzyme levels in all four strains. Ni decreased glutathione reductase in B4, slightly increased it in Ni-Tol and Cu-Tol, and did not affect the low levels of this enzyme in UTEX72. Ni lowered glutathione peroxidase in B4 and either did not affect it or slightly raised it in the other strains. Ni lowered catalase in B4 and did not affect the other strains. Superoxide dismutase was raised in B4 and Ni-Tol and lowered in Cu-Tol and UTEX72, and glucose-6-phosphate dehydrogenase was lowered in all four strains. These results suggest that one major mechanism of Ni resistance, especially in strain B4, may be the ability to combat the formation of ROS when exposed to this metal, likely by maintaining a high GSH:GSSG ratio.     

Mercury pretreatment selects an enhanced cadmium-accumulating phenotype in<Emphasis Type="Italic"> Euglena gracilis</Emphasis>

Pre-treatment of heterotrophic cultures of Euglena gracilis with 1.5 microM HgCl(2) for at least 60 generations resulted in a cell population that showed both increased resistance to Cd(2+) and ability to accumulate it, when compared to non-Hg(2+)-pretreated Euglena. These Hg(2+)-enhanced capacities were evident in cells cultured in the dark in a medium with lactate, but not in cells cultured with glutamate plus malate. After culturing with 0.1 mM CdCl(2) through three consecutive transfers, the mercury-pretreated cells still grew and maintained high levels of glutathione-related metabolites, while the non-Hg(2+)-pretreated cells died. Cultures of Hg(2+)-pretreated cells, after transfer to media with or without cadmium, did not alter either their enhanced Cd(2+) accumulation or their increased production of glutathione-related metabolites. These observations suggested that the Hg(2+)-pretreated population underwent a permanent change that improved its Cd(2+) resistance. Several factors that contributed to the improved capacities included: (a) higher cellular malate, cysteine and glutathione levels induced by Hg(2+) before and after Cd(2+) exposure; and (b) increased storage of Cd(2+) in mitochondria along with increased intramitochondrial citrate, cysteine, and glutathione levels. These characteristics suggested that this Cd(2+) hyper-accumulating strain of E. gracilis might be a suitable candidate for Cd(2+)-bioremediation of polluted water systems.     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

Regulation of phytochelatin synthesis by zinc and cadmium in marine green alga,Dunaliella tertiolecta

Although Cd(2+) is a more effective inducer of phytochelatin (PC) synthesis than Zn(2+) in higher plants, we have observed greater induction of PC synthesis by Zn(2+) than Cd(2+) in the marine green alga, Dunaliella tertiolecta. To elucidate this unique regulation of PC synthesis by Zn(2+), we investigated the effects of Zn(2+) and Cd(2+) on the activities of both phytochelatin synthase (PC synthase) and enzymes in the GSH biosynthetic pathway. PC synthase was more strongly activated by Cd(2+) than by Zn(2+), but the difference was not very big. On the other hand, gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS) were activated by both heavy metals, but their activities were higher in Zn-treated cells than in Cd-treated cells. Dose-dependent stimulation of intracellular reactive oxygen species (ROS) production was observed with Zn(2+), but not Cd(2+) treatment. These results suggest that Zn(2+) strongly promotes the synthesis of GSH through indirect activation of gamma-ECS and GS by stimulating ROS generation. This acceleration of the flux rate for GSH synthesis might mainly contribute to high level PC synthesis.     

In vitro and in vivo studies on oxygen free radical and DNA adduct formation in rat lung and liver during benzo[a]pyrene metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.     

Inhibition of cadmium-induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd(2+)) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS-supporting cells, are resistant to Cd(2+)-induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl(2) for 24 h at concentrations higher than 20 microM substantially induced astrocytic cytotoxicity, which also resulted from long-term exposure to 5 microM of CdCl(2). Intracellular calcium levels were found to rapidly increase after the addition of CdCl(2) into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd(2+)-treated astrocytes. Adenovirus-mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd(2+)-exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd(2+) exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd(2+)-induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd(2+)-primed astrocytes. This information provides a useful approach for treating Cd(2+)-induced CNS neurological disorders.     

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9mM Cd(NO(3))(2).4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination.     

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.     

Evasion of Legionella pneumophila from the bactericidal system by reactive oxygen species (ROS) in macrophages

We demonstrated that the production of reactive oxygen species (ROS) by U937 macrophage-like cells was suppressed upon infection with a wild type Legionella pneumophila strain, whereas such suppression was not observed in the case of infection with intracellular growth-deficient mutants. This was supported not only by measuring ROS released into the supernatants of cell cultures by chemiluminescence assaying but also by detecting intracellular ROS with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), under a confocal laser scanning microscope. Furthermore, more than 60% of the phagosomes containing intracellular growth-deficient mutants were colocalized with p47(phox), which is the cytosolic subunit of NADPH oxidase, consistently throughout the observation period in an early stage of bacterial infection. In contrast, the colocalization of p47(phox) was suppressed after infection with the wild type strain. These results suggest that the interference with ROS production by U937 cells infected with wild type L. pneumophila is due to a failure of NADPH oxidase activation through inhibition of p47(phox) recruitment to phagosomes harboring bacteria. The results also highlighted the difference in the nature of phagosomes between ones harboring the wild type and ones the intracellular growth-deficient strains.     

Differential interaction of maize root ferredoxin:NADP(+) oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins

In higher plants ferredoxin (Fd):NADP(+) oxidoreductase (FNR) and Fd are each distributed in photosynthetic and non-photosynthetic organs as distinct isoproteins. We have cloned cDNAs for leaf FNR (L-FNR I and L-FNR II) and root FNR (R-FNR) from maize (Zea mays L.), and produced recombinant L-FNR I and R-FNR to study their enzymatic functions through kinetic and Fd-binding analyses. The K(m) value obtained by assay for a diaphorase activity indicated that R-FNR had a 10-fold higher affinity for NADPH than L-FNR I. When we assayed for NADPH-cytochrome c reductase activity using maize photosynthetic Fd (Fd I) and non-photosynthetic Fd (Fd III), the R-FNR showed a marked difference in affinity between these two Fd isoproteins; the K(m) for Fd III was 3.0 microM and that for Fd I was 29 microM. Consistent with this, the dissociation constant for the R-FNR:Fd III complex was 10-fold smaller than that of the R-FNR:Fd I complex. This differential binding capacity was confirmed by an affinity chromatography of R-FNR on Fd-sepharose with stronger binding to Fd III. L-FNR I showed no such differential interaction with Fd I and Fd III. These data demonstrated that R-FNR has the ability to discriminate between these two types of Fds. We propose that the stronger interaction of R-FNR with Fd III is crucial for an efficient electron flux of NADPH-FNR-Fd cascade, thus supporting Fd-dependent metabolism in non-photosynthetic organs.     

Isolation and Identification of Camptothecin from Cells of Camptotheca acuminata Suspension Cultures

Ten strains of non-sulfur purple photosynthetic bacteria were isolated from soil and water samples gathered in Bangkok and its surrounding area. The isolated strains from Thailand were divided into two groups, Al to A4 and BI to B6. They were identified as Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides, respectively. All strains grew well either at 30°C or 40°C, but failed to grow at 45°C. Strains belonging to group A had weak activities of nitrogenase (acetylene reduction) and hydrogen production, while strains of group B showed much higher activities than group A. The activities of nitrogenase and hydrogen production of isolates in Thailand were compared with those of isolates in Japan. The activities of isolated strains in Thailand at 40°C were almost equal to those at 30°C or even higher. On the other hand, both hydrogen production and the nitrogenase activity of isolates in Japan decreased significantly at 40°C as compared to the activities at 30°C. These results suggest an intrinsic thermostability in hydrogen production by the non-sulfur purple photosynthetic bacteria of Thailand. Among isolated strains in Thailand, strain B5 was the most active in nitrogenase and hydrogen production, and its activity was significantly higher than strain TN3 at 40°C. TN3 had been selected as the most active strain among isolates in the Sendai area.     

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2‘,7‘-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide(PI), indicated that As2O3 (2μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells‘ susceptibility to apoptosis by As2O3.     

Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.