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.

Evaluation of mutagenic activity of the organo-selenium compound Selol by use of the Salmonella typhimurium mutagenicity assay.

We examined the mutagenic activity of the anti-oxidant Selol, an organo-selenium compound, by use of the Salmonella typhimurium mutagenicity assay (Ames test) with strains TA97a, TA98, TA100, TA 1535 and TA102 in the absence and in the presence of metabolic activation with an S9 fraction from Aroclor-induced rat liver. Doses were 330, 500, 1000 and 5000 microg per plate. Selol contains the element selenium (valency, +4) in its structure and it may have chemopreventive and anticancer activity. Selol was found to be non-toxic and non-mutagenic for test doses up to 5% per plate (which designates the declared content of Selenium (+4) as 5000 microg per plate) in all the S. typhimurium strains.

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts.

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH(total)/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 microM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100-150 microM). GSH(total)/GSSG ratio in the 100 microM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 microM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH(total)/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, L-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH(total)/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts.

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40-50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50-100 microM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl-L-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by L-buthionine-(S,R)-sulfoximine (BSO).

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.

Arginase isoforms in human colorectal cancer.

Arginase (EC 3.5.3.1) activity was determined in 54 colorectal tissues obtained from patients with primary colorectal adenocarcinoma as well as in serum of 45 patients and 65 healthy individuals. In patients, the preoperative values of the mean serum arginase activity and the activity in colorectal tumors were much higher than in serum of healthy subjects and control tissues. Two isoforms of arginase, anionic and cationic, were identified in colorectal tissues (normal and cancerogenous), and only one, the cationic form, in serum. These arginases were different from the main human liver cationic arginase (pI 9.3). The anionic colorectal arginase was identical with the human liver anionic isoform (pI 7.7), and the cationic arginase from colorectal tissues and blood serum with the human kidney cationic enzyme (pI 8.9). The total activity and the level of protein of the cationic arginase in colorectal cancer was higher than in control tissue, and it was also higher in serum of patients with colorectal cancer than in healthy subjects. Thus, it can be concluded that the increased arginase activity in blood serum and colorectal cancer in studied patients was due to the raised level of the cationic arginase and this isoform seems to be a discriminating parameter for assessing the presence of colorectal cancer.

Daminozide--lack of the genotoxic activity in the short-term bacterial tests.

Daminozide [ALAR] a plant growth regulator has been widely used on apples since the late 1960s. It has been identified as a possible carcinogen. Restrictions were ordered to reduce both application rates and allowable daminozide residue levels. Since conclusive scientific data necessary to characterize the risk of daminozide were not available, additional information on mutagenic activity of this compound was needed.

Bacterial mutagenicity of dithiocarbamate fungicide thiram.

In the present work, within a project of re-evaluation of authorized pesticides coordinated by PZH (National Institute of Hygiene) we aimed at looking for a mechanism of induction of chromosomal aberrations by thiram. We checked its ability to damage bacterial DNA.

[Selected short-term bacterial and eukaryotic tests used for detection of genotoxicity of environmental chemical contaminants]. / Wybrane krótkoterminowe testy bakteryjne i na organizmach eukariotycznych, stosowane do oceny genotoksycznosci srodowiskowych zanieczyszczen chemicznych.

During the last years human exposure to environmental chemical contaminants (mutagenic/carcinogenic agents) has greatly increased. Evaluation of the biological effect of human exposure to mutagenic/carcinogenic agents in short-term tests is very important. In present paper the bacterial and eukaryotic tests for detection of genotoxic effect of environmental chemical contaminants have been described.

The effects of captan and captafol on different bacterial strains and on c-mitosis in V79 Chinese hamster fibroblasts.

The mutagenic activity of captan and captafol was tested using Ames strains and strains showing an SOS response. Captafol was mutagenic in S. typhimurium strain TA102 (uvr+) and captan in strain TA104 (uvrB). Both captan and captafol elicit damages in DNA recognized by correndonuclease II, as shown by the repair test, and induced the SOS repair system in E. coli PQ37 (uvrA) strain. Only captafol induced the SOS system in PQ35 (uvr+). The lack of induction of beta-galactosidase at nonpermissive temperature in E. coli MD332 (dnaCs uvrA) strain showed that neither chemical was able to produce DNA breaks. In V79 Chinese hamster fibroblasts higher induction of c-mitosis by captafol than by captan (22% and 15% over the control, respectively) was accompanied by a higher decrease in nonprotein sulfhydryl groups, mainly GSH (41% and 77%, respectively). The content of protein sulfhydryl groups was decreased by either fungicide to a similar extent.

Isolation and studies of the mutagenic activity of saponins in the Ames test.

Mutagenic activity of medicagenic acid, medicagenic acid 3-0-glucopyranoside and soyasaponin I was tested by the Ames method with S. typhimurium strains TA97, TA98, TA100, TA102 in the absence and the presence of metabolic activation (S9 mix). These saponins have been isolated and identified from alfalfa roots and clover Trifolium incarnatum seeds. All of them were found to be non-toxic and non-mutagenic for testing doses.

Mutagenicity of carboline methyl derivatives in Salmonella.

A series of linear, methyl-substituted derivatives of 5H-indolo[2,3-b] quinolines was tested for mutagenic activity with the battery of Ames tester strains. Mutagenic activity of indoloquinolones was strongly influenced by the position and a number of methyl groups. All compounds tested, with one exception, act like frame-shift mutagens. Only two compounds among them were mutagenic in the strain detecting oxidative and cross-linking mutagens.

cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase.

A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of O6-methylguanine residues. Following three exposures to N-methyl-N'-nitro-N-nitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated. Extracts of BK2210 cells hosting the RMGMT plasmid expressed a O6-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells. The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68% homology with the human transferase.

Screening for genotoxic activity of amitraz with short-term bacterial assays.

Genotoxic effects of both amitraz and its metabolites made by S9 fraction were reevaluated in short-term bacterial assays. Neither amitraz nor its metabolites induced frameshift mutation or caused base-pair substitution as detected by the Ames test. They also did not introduce any damages into DNA recognized by correndonuclease II as shown by the repair test. Metabolites of amitraz (but not amitraz itself) induced the SOS-repair system in E. coli strain PQ 243 tagA, alkA which was deficient in N-glycosylases. It is concluded that neither amitraz nor its metabolites have mutagenic activity. In contrast to amitraz, its metabolites alkylate DNA in the N3-position of adenine.

Molecular forms of alkaline phosphatase in particular segments of the small intestine of adult rat. Separation of three enzyme forms from jejunum.

Rat duodenum and jejunum were found to contain three forms of alkaline phosphatase, F1, F2 and F3, and ileum two forms of this enzyme, F1 and F2. The procedure for separation of phosphatase F1, F2 and F3 from jejunum is presented.

Effect of unilateral nephrectomy on the RNA synthesis in hypertrophic rat kidney.

1. The hypertrophic rat kidney after unilateral nephrectomy showed a time-dependent increase in the RNA/DNA ratio. This was associated with the increased rate of alpha-amanitin-insensitive RNA synthesis in isolated nuclei. 2. The increased activity of solubilized RNA polymerase was due to polymerase I, II and III as judged from the DEAE-Sephadex chromatography. 3. Chromatin DNA from hypertrophic kidneys was more susceptible to DNAase I than DNA from control kidneys.