Maria Szumiło

Induction of rat liver cytochrome P450 isoenzymes CYP 1A and CYP 2B by different fungicides, nitrofurans, and quercetin.

The genotoxic activity of environmental xenobiotics is manifested either in their direct interaction with cellular genetic material or in provoking secondary events, among which reactive oxygen species (ROS) production is a common phenomenon. Both pathways can be mediated by the activity of the cytochrome P450 monooxygenase system. We studied induction of the CYP 1A or CYP 2B monooxygenases in rat liver by the fungicides: thiram, captan, captafol, dodine and the drugs: nitrofurazone, furazolidone and the plant flavonoid: quercetin. A cytochrome P450 induction assay (CYPIA test) was used. S9 prepared from livers of rats treated with the test compounds were used to activate ethidium bromide (EtBr) (CYP 1A isoenzyme) or cyclophosphamide (CPA) (CYP 2B isoenzyme) in the Ames test. It was found that among the tested compounds, the most potent inducer of CYP 1A was furazolidone (3 x 80 mg/kg). Less potent was thiram (1 x 100mg/kg), as well as quercetin (3 x 80 mg/kg), and captafol (1 x 30 mg/kg). On the other hand, thiram (1 x 100 mg/kg), captafol (1 x 30 mg/kg), and quercetin (3 x 80 mg/kg) were most potent in the CYP 2B isoenzyme induction, while furazolidone (3 x 80 mg/kg), and nitrofurazone (3 x 80 mg/kg) appeared to be less potent in this respect. Captan and dodine (3 x 80 mg/kg) did not affect the activity of any of the cytochrome P450 isoenzymes.

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS--thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).

Protective effect of N-acetyl-L-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells.

This work investigated the effect of N-acetyl-L-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 μM concentration. It was evidenced by a statistically significant increase of both GSH(t) and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH(t) level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.

Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells

Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action.

Modulation of antioxidant defense system by the dithiocarbamate fungicides Maneb and Zineb in Chinese hamster V79 cells and the role of N-acetyl-L-cysteine.

Oxidative stress is one of the major factors leading to Maneb- and Zineb-induced disorders. The aim of this in vitro study was to examine (i) the potency of Maneb and Zineb to induce changes in antioxidant enzyme activities in Chinese hamster fibroblasts V79 cells and (ii) the role of N-acetyl-L-cysteine (NAC) in the preventing their action. Maneb increased mitochondrial superoxide dismutase (SOD2) activity but failed to affect the activity of cytoplasmic superoxide dismutase (SOD1), whereas Zineb did not change the activity of any of superoxide dismutases. The activity of catalase (CAT) was reduced only by Zineb. The activity of both glutathione peroxidases (non-Se-GPx, Se-GPx) and glutathione reductase (GR) was decreased after exposure to these agents. After NAC pre-treatment Maneb increased the activity of GR, whereas the activity of non-Se-GPx was decreased as compared to that in NAC-treated cells. On the other hand, the activity of both SODs and CAT was decreased. Zineb decreased the activity of both GPxs and SOD2 with a concomitant increase in CAT activity comparing to NAC-treated cells. The results obtained thus suggest that Zineb acts by another mechanism, than Maneb does, and that one of the mechanisms of NAC protection against Maneb or Zineb-induced effects in V79 cells is its impact on enzymatic defense. Activity of GR, SOD2, and GPxs are the most affected enzymes.

The effects of sodium diethyldithiocarbamate in fibroblasts V79 cells in relation to cytotoxicity, antioxidative enzymes, glutathione, and apoptosis

Sodium diethyldithiocarbamate (DETC) is the main metabolite of disulfiram. Recently, we reported that mechanism of disulfiram cytotoxicity in V79 cells might be partially connected with thiol redox-state imbalance. Here, we examined the effect of DETC on the level of intracellular glutathione (GSH), protein oxidation (measured as PC—protein carbonyl content), lipid peroxidation (measured as TBARS—thiobarbituric acid reactive substances), antioxidant enzymatic defense, as well as on apoptosis. We used V79 Chinese hamster fibroblasts cells with and without modulated glutathione (GSH) level by N-acetyl-l-cysteine (NAC). We showed that treatment with DETC at concentrations that cause a moderate increase in thiol-state imbalance but not cell death stimulates oxidative stress measured as increased level of PC and TBARS, adaptive response of GSH-related enzymes and apoptosis. Our results show that cellular effects of DETC are partially attributable to the initial redox cellular state, since the increase of GSH level by NAC pre-treatment prevented the observed changes.

Selol (Se IV) modulates adhesive molecules in control and TNF-α-stimulated HMEC-1 cells

Selol, an organic selenitetrigliceride formulation containing selenium at +4 oxidation level, has been suggested as anticancer drug. One of the causes of several diseases including cancer may be inflammation. This study aimed at determining the activity of Selol via measuring its effect on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-κB) activation, intercellular cell adhesion molecules-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1), and plateled-endothelial cell adhesive molecule-1 (PECAM-1) levels on control and on tumor necrosis factor-α (TNF-α)-stimulated human microvascular endothelial cells (HMEC-1). Cells were treated either with Selol 5% (4 or 8 μgSe/mL) or TNF-α (10 ng/mL) alone or with Selol concomitant with TNF-α. Selol treatment resulted in ROS generation, activation of NF-κB, downregulation of PECAM-1, VCAM-1 and slight upregulation ICAM-1 expression on the cell surface. TNF-α treatment reflected in sharp NF-κB activation, upregulation of both ICAM-1 and VCAM-1 in parallel with the downregulation of PECAM-1 expression on cell surface. Exposure to both compounds upregulated ICAM-1 and VCAM-1, downregulated PECAM-1 level on cell surface in parallel with no changes in level of NF-κB activation as compared with effects mediated by TNF-α alone. These results points to new look at Selol action since it shows a pro-inflammatory activity in parallel with effects on CAMs expression on the cell surface of human microvascular endothelial cells. However, since Selol enhances CAMs expression level when is present concomitantly with TNF-α this fact might suggest that selenium present in the condition of inflammation will make it worse.