DNA adducts and liver DNA replication in rats during chronic exposure to N-nitrosodimethylamine (NDMA) and their relationships to the dose-dependence of NDMA hepatocarcinogenesis

Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.     

Concentration of nitrosodimethylamine in commercial pelleted experimental animal diets

To know the conditions of contamination by the chemicals in the experimental animal diets, the contents of nitrosodimethylamine (NDMA) were analyzed in the diets for mice and rats. The raw materials of the diets were also analyzed. In addition, NDMA levels were determined in the diets which were stored long or heated in the autoclaves. The averages of NDMA in these diets were less than 10 ppb. In the raw materials, alfalfa contained a higher levels of NDMA. There was no clear tendency of increase or decrease in NDMA levels, when the diets were stored for six months at room temperature (23-25 degrees C) or at low temperature (7-10 degrees C). Heating the diets at 121 degrees C, no remarkable changes were observed in NDMA levels. It seems that little adverse influences on the conduct of the animal experiments would be anticipated by the NDMA in the commercial diets.     

Operculina turpethum attenuates N-nitrosodimethylamine induced toxic liver injury and clastogenicity in rats

The root extract of Operculina turpethum (OTE) has been used as an anti-inflammatory, purgative, and hepato-protective agent. N-Nitrosodimethylamine (NDMA) is a potent hepatotoxin that induces fibrosis of the liver. In the present study, we examined the therapeutic effects of OTE root extract against NDMA-induced hepatotoxicity and clastogenicity in rats. Hepatic fibrosis was induced in adult male albino rats through serial intraperitoneal administrations of NDMA at a concentration of 10 mg/kg body weight on three consecutive days of each week over a period of three weeks. A group of rats received OTE orally in doses of 75, 150 and 200 mg/kg body weight at 5 h after the administration of NDMA. The controls and treated animals were sacrificed on days-7, 14 and 21 after the start of the administration of NDMA. The progression of hepatic fibrosis as well as the amelioration effect of OTE was evaluated through histopathologically as well as by immunohistochemical staining for the activation of hepatic stellate cells. Alterations in serum and liver biochemical parameters and LDH isoenzymes were also studied. Serial administration of NDMA resulted in well formed fibrosis in the liver and induction of micronuclei in the bone marrow cells. Staining of α-SMA demonstrated activated stellate cells from day-7 onwards which was dramatically increased on day-21. An elevation of micronuclei count, liver function enzymes, serum hydroxyproline levels and LDH isoenzymes 4 and 5 were also observed. All these changes were remarkably reduced in OTE administered animals and fibrogenesis was completely absent. Our results suggest that OTE has hepatoprotective and anti-clastogenic effects against NDMA-induced hepatic fibrosis. Therefore OTE may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.     

Betulinic acid protects against N-nitrosodimethylamine-induced redox imbalance in testes of rats

Objectives: N-nitrosodimethylamine (NDMA) is known to elicit carcinogenic activity in the liver and kidney of animals. There is a dearth of information of its effect in testis. This study evaluated the protective role of betulinic acid (BA) against NDMA-induced redox imbalance in testes of rats.

Methodology: Twenty-four male rats were assigned into four groups and treated with normal saline, BA, NDMA and [BA+NDMA]. BA (25 mg/kg) was given for 14 days, while NDMA (5 mg/kg) was given on days 7 and 12.

Results: Administration of NDMA significantly increased the weight and relative weight of testes by 51 and 71%, respectively, while treatment with BA attenuated the weight-gain. Furthermore, NDMA decreased the sperm count, motility and live–dead ratio by 57, 36 and 37%, respectively, and increased total sperm abnormality by 56%. However, BA attenuated the changes in the spermiogram of NDMA-treated rats. NDMA significantly decreased the activities of antioxidative enzymes, follicle-stimulating and luteinizing hormones, while testicular levels of thiobarbituric acid reactive substances and total cholesterol were increased. Also, NDMA increased the activities of aniline hydroxylase and aminopyrine-N-demethylase. Supplementation with BA attenuated NDMA-induced alteration in these biochemical indices.

Conclusion: BA protects against NDMA-induced redox imbalance via activation of antioxidative pathway.     

Mitotic cycle of a HeLa cell culture exposed to the maximum permissible concentrations of trace elements

It is shown that administration of certain trace elements in maximum allowable concentrations induces changes in metabolism and functionation of cells in the culture. Zinc, nickel, cobalt, cadmium and fluorine are stated to inhibit mitotic activity of HeLa cells by the end of 24 hours of their action. Parallel with this they promote a decrease in H3-thymidine incorporation into DNA. Besides these substances delay various stages of the mitotic cycle for cells.     

Alterations in membrane permeability of malaria-infected human erythrocytes are related to the growth stage of the parasite

During the intraerythrocytic growth of Plasmodium falciparum in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.     

Decrease of <Emphasis Type="Italic">N</Emphasis>-nitrosodimethylamine and <Emphasis Type="Italic">N</Emphasis>-nitrosodiethylamine by <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> R3 is associated with surface-layer proteins

The objective of this study was to evaluate the ability of five strains of meat-borne bacteria to decrease N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) and to elucidate the mechanism in Mann-Rogosa-Sharp (MRS) broth. Lactobacillus pentosus R3 was found to be the most effective in decreasing the concentration of the two N-nitrosamines (NAs) in MRS broth, with a rate of 22.05% for NDMA and 23.31% for NDEA. The concentration of the two NAs could not be reduced by either extracellular metabolites or intracellular extracts of Lb. pentosus R3 (P?>?0.05), and proteinaceous substances in the cell debris were found to be responsible for the decrease. These were surface-layer proteins (SLPs) located on the cell wall. Therefore, the decrease in NDMA and NDEA by Lb. pentosus R3 is associated with its SLPs. Lb. pentosus R3 may be developed as a starter culture in the production of fermented foods with lower NAs.     

About mechanisms of formation of multinuclear hepatocytes during toxic action of N-nitrosodimethylamine on rats

The processes of hepatocyte multinucleation were studied in rats exposed to N-nitrosodimethylamine (NDMA). Using the immunohistochemical reaction to γ-tubulin, it was established that the number of cells containing three or more centrosomes increased 48 h after the NDMA injection. The formation of additional centrosomes in hepatocytes was shown to be based on the oxidative stress induced by NDMA metabolism with the participation of the cytochrome P450 superfamily. The administration of NDMA led to a sharp increase in the cytochrome P450 content in liver, especially 24 and 48 h (3.3 and 2.8 times, respectively) after the NDMA injection. The immunohistochemical reaction for cytochrome P4502E1 revealed an intensive staining of the cytoplasm of centrilobular hepatocytes 24 and 48 h after the NDMA action. In the same time period, a 1.1-2.0-fold increase occurs in the concentration of malonic dialdehyde (MDA) (a derivative of lipid peroxidation) and a 1.1-1.3-fold decrease in catalase activity (an enzyme of the cell antioxidative system). At a later time (72–120 h) after the NDMA action, the number of cells with three or more centrosomes, the intensity of cell cytoplasmic staining for cytochrome P450 2E1, and the concentrations of P450 and MDA in the liver decreased, whereas catalase activity increased. After 48 h of NDMA treatment, the incorporation of binuclear hepatocytes with various ³H-thymidines into nuclei occured, which indicates asynchronous DNA synthesis. The immunohistochemical reaction for pKi-67, nuclear protein that is a marker of cell proliferation, has established that the asynchronicity of nuclear proliferative activity in binuclear cells is not only characteristic of the S phase, but also of other cell cycle phases, including G₁, G₂, and M. Thus, the main mechanisms of hepatocyte multinucleation under the influence of NDMA are as follows: (1) increased hyperamplification of centrosomes as a consequence of oxidative stress and (2) asynchronous DNA synthesis in nuclei of binuclear hepatocytes with subsequent asynchronous acytokinetic mitosis.     

Influence of the physiologically widespread substances on the oxidative activity of copper(II) complexes with sinefungin, a nucleoside antibiotic

The oxidation-promoting reactivity of the Cu(II)-sinefungin complex in the presence of hydrogen peroxide was studied at pH 7.4, using N,N-dimethyl-p-nitrosoaniline (NDMA), as well as plasmid DNA as target molecules. Mixture of the complex with H(2)O(2) was found to be an efficient oxidant, bleaching NDMA solution, and generating single- and double-strand breaks in DNA. The oxidative DNA damage was investigated also in the presence of varying amounts of glutathione, histidine, Gly-Gly-His peptide, H2A histone, and ascorbic acid, showing diverse influence of those substances on the cleavage extension.     

Changes in the content of phenolic substances during the growth ofNicotiana tabacum cell suspension culture

The dynamics of changes in the content of four groups of phenolic substances was investigated during the growth cycle of the cell suspension culture ofNicotiana tábacum by means of fractionation. The relative contents of free phenolic acids, their esters, phenolic glycosides, and phenole acids non-extractable with methanol changed in dependence on the growth phase of the culture. A sharp increase, especially in the content of ester- and glycoside-bound phenolics and to a lesser extent also of phenolics belonging to the other two groups, occurred at the end of the lag phase. Then, after a temporary decrease at the early linear phase, the level of phenolics in the three fractions representing bound forms considerably increased again at the late linear and early stationary phases. The synthesized phenolic substances were partially released from the cells into the cultivation medium, which contained 15 to 30 % of the total content of the phenolics in the culture at different phases of the growth cycle. Likely causes of these changes are discussed.     

Biotransformation enzyme-dependent formation of micronucleus and multinuclei in cell line V79-hCYP2E1-hSULT1A1 by 2-nitropropane and N-nitrosodimethylamine

V79-hCYP2E1-hSULT1A1, a V79-derived cell line co-expressing both human CYP2E1 and SULT1A1, has been constructed and efficiently used in detection of the mutagenic activities of a number of promutagens. 2-Nitropropane (2-NP) and N-nitrosodimethylamine (NDMA), both being hepatocarcinogenic to animals but inactive in standard genotoxicity assays in vitro, are activated to mutagenic metabolites by human SULT1A1 and CYP2E1, respectively. Nevertheless, little is known about the chromosomal effects of these two carcinogens. In the present study, we investigated the effects of 2-NP and NDMA on frequencies of micronucleated (F(mi)) and multinucleated cells (F(mu)) in V79-hCYP2E1-hSULT1A1 cells. The results showed induction of both F(mi) and F(mu) by 2-NP and NDMA individually, and this effect was completely suppressed by relatively specific inhibitor of SULT1A1 and CYP2E1, i.e., pentachlorophenol and 1-aminobenzotriazole, respectively. The F(mu)/F(mi) ratio in 2-NP groups was significantly higher than NDMA groups, probably indicating an aneugenic activity of 2-NP based on proposed F(mu)/F(mi) ratio as a simple index to discriminate aneugens from clastogens. The present study has established biotransformation enzyme-dependent formation of multinuclei and micronuclei induced by 2-NP and NDMA.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

Subinhibitory concentrations of organic ammonium salts: The effect on biological properties ofSalmonella typhimurium

The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells. The culture filtrates were prepared by 1-d cultivation ofSalmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37°C after addition of subinhibitory concentrations of organic ammonium salts. The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells. The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10⁶ PFU/mL. SubstancesC3, C9 andC12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs. One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substanceA16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.     

Phenolic substances in the cell suspension culture ofCentaurium erythraea

The content of phenolic substances in the cell suspension culture ofCentaurium erythraea fluctuated during a 21-day-long subcultivation period in dependence on the growth phase. The total relative content of the phenolics reached its maximum at the time of transition to the exponential growth phase, similarly as the fraction of free phenolic acids, glycosides, and the fraction of phenolic acids released from the cells after alcaline hydrolysis. On the other hand, the content of phenolic acid esters decreased at this growth phase of the culture. Changes in the level of phenolic substances in the culture medium corresponded in their character to changes in the relative content of the phenolics in the cells.     

The reaction of the neuroblastoma cells in the culture on the influence of tretionine and neurotoxine

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.     

C3H/He Mice as an Incompatible Cholangiocarcinoma Model by Clonorchis sinensis,Dicyclanil and N-Nitrosodimethylamine

Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm³) from the Cs group was a hepatocellular adenoma and the other (280.6 mm³) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.     

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure.     

Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Characteristics of action of precursors of carcinogenic nitroso-compounds on cell cultures

On the primary rat lung cell cultures, a study was made of the transforming action of sodium nitrite (NN) and amidopyrine combination with the control for formation of carcinogen--N-nitrosodimethylamine (NDMA) in growth medium. The manifestation of transformation was registered by appearance of morphological changes and multilayer growth foci. As criteria for evaluation after 48 hour treatments with NDMA, NN and AP, were used DNA-synthetizing activity of cells, mitotic index, frequency of mitoses pathology, monolayer density. The effects of transforming dose of NN alone and in combination with AP were the same. But malignization (tumor development in newborn rats in the points of cell suspension inoculation) took place only after administration of NN in combination with AP, when carcinogen was formed. Theophylline decreased the action of NN-AP combination.     

Inhibitory effect of saturated fatty acids on the mutagenicity of N-nitrosodimethylamine

Saturated fatty acids, C5-C12, inhibited the mutagenic activity of N-nitrosodimethylamine (NDMA) in E. coli WP2 uvrA/pKM101. The inhibition by laurate (C12) was due to the suppression of the enzymatic demethylation of NDMA, whereas that by caprate (C10) was simply due to the bactericidal effect of the fatty acid. Caproate (C6) did not affect the NDMA-demethylase, and evidence is presented to show that the inhibition of mutagenesis by caproate was a result of its interference with the uptake of NDMA metabolites into bacterial cells. Possible biological significance of the inhibition is discussed.