Repair studies on methylated DNA of CHO cells.

DNA methylation pattern was investigated on Chinese hamster ovary (CHO) cells after treatment with N-(14C)-methyl-N-nitrosourea (14C-MNU). The main target was the N-7 position of guanine, exceeding the methylation in the O6 position of guanine by a factor of 8 and that in the N-3 position of guanine and adenine by a factor of 20. No DNA repair could be observed within 2 hours after methylation. Pretreatment of cells with gamma irradiation (7 rad) before application of MNU induced repair of N-7-methylguanine. This methylation product was decreased to about 50% within two hours, whereas the repair of the other methylated bases was not influenced. The analysis was carried out by high performance liquid chromatography after acid hydrolysis of isolated DNA. 14C-methylated products were determined by liquid scintillation counting.

Mutagenicity of chlorinated polycyclic aromatic compounds.

The catalytic chlorination of polycyclic aromatic hydrocarbons leads to the formation of complex mixtures of isomers analogous to those found in fly ash samples of an incineration plant for radioactive waste. Therefore chlorination mixtures of chrysene, pyrene, and fluoranthene were analyzed and characterized by gas chromatography-mass spectrometry. Determination of the exact position of each chlorosubstituent within the molecule was not possible either by mass spectrometry or by comparison with well-defined methyl analogs. The toxic as well as mutagenic effects of such mixtures of isomers was compared with those of their parent hydrocarbons in the microtiter fluctuation test using Salmonella typhimurium TA 98 and TA 100 in a concentration range of 0.5 to 5.0 microgram/ml. No toxic effect could be observed, but the chlorinated products in contrast to their parent compounds were found to be strong mutagens to the S. typhimurium test strains and showed a positive response even without enzymatic activation. Frameshift mutations as well as base pair alterations were detected.

Studies on the metabolic activation of diethanolnitrosamine in animal-mediated and in vitro assays using Escherichia coli K-12 343/113 as an indicator.

The mutagenic activity of diethanolnitrosamine (NDELA), a carcinogenic compound which leads to inconsistent results in standard in vitro procedures was tested in vitro and in animal-mediated assays with the indicator strain Escherichia coli (E. coli) K-12 343/113. This strain allows the simultaneous detection of forward and back mutations arising in several genes of the E. coli chromosome. In animal-mediated assays in which mice were used as hosts for i.v. injected E. coli indicator cells, s.c. application of NDELA induced a dose dependent increase of galactose fermenting mutants in cells recovered from the livers of animals exposed for 3 h to the mutagen. Comparison with results obtained with diethylnitrosamine (DENA) in the same test system revealed that the two compounds apparently cause different types of mutagenic lesions. Induction of arg+ mutations by DENA and several other aliphatic nitrosamines is mainly due to base pair substitutions, whereas NDELA is rather mutagenic in the galRs system. This latter system is, in addition, sensitive to frameshifts and deletions. These differences in mutagenic specificity suggest that NDELA and DENA, although structurally closely related, are activated via different molecular mechanisms. In fact, evidence is accumulating that alcohol dehydrogenase (ADH) could be involved in the activation of NDELA. On the other hand, the effective mutagenesis of NDELA obtained in vitro with E. coli upon addition of rat liver microsomal fraction would not be expected if ADH is involved in the activation since the S-9 Mix used in the present experiments was devoid of cofactors (NAD, NADP), necessary to accomplish oxidation by ADH. Therefore, further in vivo studies were performed, in which pyrazole, a potent blocker of ADH, was administered prior (1 and 24 h) to the injection of the mutagen. The observation that a dose dependent increase of mutants in the liver (and to a lower extent in the spleens) of treated animals takes place under conditions in which ADH activity is blocked, whereas several microsomal enzymes are stimulated, indicated that besides oxidation of NDELA by ADH other metabolic activation pathways are involved. Apparently enzymes contained in the liver homogenate, possibly NADPH dependent enzymes of the microsomal ethanol oxidizing system, play an important role in the formation of mutagenic metabolites of NDELA.

Concentration of dimethylnitrosamine in the air of smoke-filled rooms.

In order to evaluate the contribution of volatile nitrosamines from tobacco smoke to indoor air pollution, N-nitroso-dimethylamine (NDMA) and N-nitroso-diethylamine (NDEA) were measured in indoor air under artificial and natural conditions. In controlled experiments under extreme conditions, we found that tobacco smoke-related NDMA levels above 0.07 ng/liter were associated with a highly irritating atmosphere which was scarcely tolerable to those present. In smoke-filled rooms under natural conditions NDMA levels ranged from 0.02 to 0.05 ng/liter except a minimum value of less than 0.01 ng/liter in a restaurant and a maximum of 0.07 ng/liter in a dancing bar. These NDMA levels are thus below comparable values reported by others. The NDMA/NDEA ratios found in air samples taken from some rooms under conditions of everyday life are quite different from those found in sidestream smoke of cigarettes. Irritation was not reported under natural conditions. From the results it is concluded that NDMA levels, measured under real life conditions, are usually not caused by tobacco smoke alone. Evidence for other sources of volatile nitrosamines is discussed.

Improved method for routine determination of nicotine and its main metabolites in biological fluids.

ExtrelutR extraction and glass capillary gas chromatography were applied to the routine determination of nicotine and its metabolites cotinine, nicotine-1'-N-oxide and cotinine-1-N-oxide in urine and plasma. After extraction of nicotine and cotinine both N-oxides and phendimetrazine-N-oxide (used as internal standard) were reduced to their bases by SO2 on-column and eluted by a mixture of diethyl ether and dichloromethane. The minimum detectable concentrations are 0.03 micrograms/ml for urinary nicotine and cotinine and 0.1 microgram/ml for the N-oxides. In plasma samples the corresponding values are 5 ng/ml and 15 ng/ml, respectively, with sample values as small as 2 ml. The advantage of the direct determination of all four compounds of interest in one sample reduced the amount of plasma required. The straightforward and rapid extraction and reduction procedure as well as the long-term stability of the gas chromatographic separation system make the method suitable for routine application.

Increased concentration of 7-methylguanine and 1-methylhypoxanthine in urine of rats bearing Yoshida tumour.

Sprague-Dawley rats bearing Yoshida Tumour showed an increased excretion of 7-methylguanine and 1-methylhypoxanthine with their urine as compared to control animals. The concentration of these methylated purines, which belonged to the non-specific cancer markers, was already significantly higher 2 days after tumour injection and more than 2-fold after 3 additional days. This was proved by daily L-[14CH3]methionine supply. Separation and identification of these compounds was done by liquid chromatography and mass spectrometry. By RNA analysis it became evident that tumour RNA contained more methyl groups than liver RNA of control rats. This suggested that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to an increased turnover of tumour RNA but also to its higher degree of methylation.

[Methylation of nucleic acids and its relation to carcinogenesis (author's transl)]. / Die Methylierung von Nukleinsäuren und ihr Zusammenhang mit der Karzinogenese.

The physiological methylation of nucleic acids and its relation to their biological function are discussed and compared with the methylation which occurs during chemical, viral and radiation carcinogenesis. The significance of methylated nucleic acid components as cancer markers is evaluated.

Metabolic studies of Hg-203 on Chlamydomonas reinhardi.

Sterile cultures of Chlamydomonas reinhardi, WT+, were treated with Hg-203 at 25 degrees C to identify probably formed volatile mercury compounds. Experiments were performed with living and dead cells under aerobic or anaerobic conditions, respectively, and the mercury concentration was measured in the system algae/nutrient medium. We found a timerelated decrease of mercury concentration in the cell suspension and the cell-free nutrient medium due to a reduction of Hg++ to Hg0, probably caused by extracellular enzymes; monomethyl or dimethyl mercury could not be detected.