Re-evaluation of the RBE of 29 kV x-rays (mammography x-rays) relative to 220 kV x-rays using neoplastic transformation of human CGL1-hybrid cells.

Neoplastic transformation of human CGL1-hybrid cells was examined after exposure to 29 kV x-rays (mammography x-rays) and conventional 220 kV x-rays. The study was designed to repeat, under well-defined irradiation and culture conditions, an earlier investigation by Frankenberg et al. (Radiat Res, 2002), and to assess the validity of the high RBE values of 29 kV x-rays that had been reported. The experiments with the two types of x-rays were performed simultaneously and shared the same controls. The transformation yields with both radiation qualities were fitted to the linear-quadratic dependence on absorbed dose, and a corresponding analysis was performed for the data earlier obtained by Frankenberg et al. The transformation yields in the present study exceed those in the earlier investigation substantially, and it appears that the difference reflects inadequate feeding conditions of the cell cultures in the early experiments. The standard error bands of the dose response curves are derived and are seen to be considerably more narrow in the present results. The lowest dose of the 29 kV x-rays was 1 Gy in both studies, and at this dose the RBE vs. the conventional x-rays has now been found to be 2 with a 95% confidence interval of 1.4-2.6. The previous result was about 3.2, but the 95% confidence is very broad for these data. The estimated limit at low doses is 3.4 in the present experiments with a confidence interval that extends from less than 2 to large values.

Genotoxicity of 4-chloro-o-toluidine in Salmonella typhimurium, human lymphocytes and V79 cells.

In the absence of a metabolizing system (S9 mix) 4-chloro-o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells. In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S. typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.

[Studies on the effect of an alkaloid extract of Symphytum officinale on human lymphocyte cultures]. / Wirkung eines Alkaloidextraktes von Symphytum officinale auf menschliche Lymphocytenkulturen.

An alkaloid extract of Symphytum officinale was investigated for its chromosome-damaging effect in human lymphocytes in vitro. In concentrations of 1.4 micrograms/ml and 14 micrograms/ml the alkaloids had no effect, in concentrations of 140 micrograms/ml and 1400 micrograms/ml the alkaloids induced sister chromatid exchanges (SCE) as well as chromosome aberrations. Additionally, the influence of rat liver enzymes (S9) was tested. The SCE-inducing capacity and the clastogenic effect of Symphytum alkaloids was increased by simultaneous application of S9-Mix.

Genotoxicity of 2-nitropropane and 1-nitropropane in Salmonella typhimurium and human lymphocytes.

A 10- and 12-fold increase of revertant numbers could be demonstrated for 2-nitropropane (2-NP of greater than 99% purity) tested in the preincubation assay with Salmonella typhimurium strains TA 100 and TA 98 in the presence and absence of S9 mix. In the nitroreductase-deficient strains TA 100NR and TA 98NR, 2-NP was less mutagenic than in the parent strains. In human lymphocytes the induction of a weak clastogenic effect and of sister chromatid exchanges required exogenous metabolic activation. No significant mutagenic or cytogenetic response was found with 1-nitropropane of 97% purity in S. typhimurium or human lymphocytes.

Dose-response relationship for chromosome aberrations induced by fecapentaene-12 in human lymphocytes.

Chromosome analyses were carried out in human lymphocytes exposed to a synthetic racemic all-trans fecapentaene-12 at 2-24 microM. A dose-dependent increase of the incidences of chromatid-type changes with distinct saturation at higher doses could be observed. The results reveal for the first time that fec-12 is a potent direct-acting mutagen in human lymphocytes.

The effectiveness of S9 and microsomal mix on activation of cyclophosphamide to induce genotoxicity in human lymphocytes.

Comparative results are presented on the effectiveness of rat-liver S9 or microsomal mix (M mix) in activating cyclophosphamide (CP) and its ability to induce a clastogenic effect in human lymphocytes in vitro. Structural chromosome changes were analysed exclusively in 1st division (M1) metaphases post-exposure. A high genotoxic response was observed for both metabolizing systems used. With an exposure of 2 h to different concentrations of S9 or M mix, the highest aberration yields were always found for the highest protein content. For CP treatment times of 1, 2 or 4 h together with S9 mix (protein content 10 mg/ml) or M mix (4 mg/ml), the latter was more efficient. With both systems, a lower clastogenic effect of CP was found at 4 h exposure than at 1 h or 2 h. Only a weak cytotoxic effect, reflected mainly by the reduction in the percentage of 3rd cycle cells (M3), and measured in terms of the proportion of M1, M2 and M3 cells, was induced by both systems.

Formaldehyde-induced cytotoxic, genotoxic and mutagenic response in human lymphocytes and Salmonella typhimurium.

The incidences of chromatid-type aberrations and sister-chromatid exchanges were significantly increased in human lymphocytes treated with formaldehyde (FA) in vitro. In the presence of the mammalian metabolic activation system, i.e. S9 mix, the yields were reduced, although not to control levels. With S9 mix the structural chromosome damage induced by exposure to 1.0 mM FA was qualitatively and quantitatively identical to that induced by 0.05 mM cyclophosphamide (used as positive control for metabolic activation). Cell proliferation was clearly reduced with or without the presence of S9 mix. In a plate assay with Salmonella typhimurium strain TA100 in the absence and presence of S9 mix, a weak mutagenic response was observed. Using the pre-incubation method, FA induced without S9 mix a 1.6-fold and with S9 mix a 2.7-fold increase of revertant numbers over controls.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. IV. Relationship between the number of his- bacteria plated and number of his+ revertants scored in the Salmonella mutagenicity test.

Five laboratories participated in a joint ring study to investigate the role of bacterial cell number in the Salmonella mutagenicity test. A strictly standardized protocol, using sodium azide and TA 1535, was developed and employed to test the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with different dilutions of Salmonella typhimurium TA-100 and TA-1535 cultures. All laboratories detected the mutagenic activity of sodium azide with only a 2-fold variation of test results. For MNNG the interlaboratory variation was approximately 5-fold. Decreasing numbers of test bacteria employed resulted in lower numbers of MNNG-induced revertants in all laboratories. The number of preexisting revertants decreased in direct proportion to the reduced cell content, whereas the number of spontaneous revertants was not as greatly affected. A critical amount of test bacteria was required in order to obtain numbers of induced revertants which were equal to twice the number of spontaneous revertants. Two evaluation parameters which may be employed to describe the mutagenicity of a compound are compared.

Threshold levels in toxicology: significance of inactivation mechanisms.

Metabolic inactivation of chemicals may prevent toxic effects of reactive intermediates when present at low levels whereas inactivation may be overcome at high levels changing dose-effect relation. This is demonstrated in various in vitro test systems: a) Monooxygenase-mediated metabolism causes formation of reactive oxygen species which induce DNA repair in lymphoblastoid cells. DNA damage is suppressed in the presence of glutathione (GSH), catalase or superoxide dismutase. b) Chloroprene is mutagenic in Salmonella typhimurium but not carcinogenic, possibly due to inactivation by GSH-conjugations. c) Chlorodinitrobenzene is not mutagenic is Salmonella typhimurium in the presence of GSH. However it is increasingly mutagenic at concentrations exceeding those of the GSH. d) Suppression of glucuronidation and sulfation in isolated hepatocytes highly increases irreversible binding of naphthalene. It is concluded that information on the metabolism of chemicals is essential for interpretation of toxicity studies in animals and their relevance to man.

Mutagenicity testing with Salmonella microsome test.

This in vitro mutagenicity test system comprises five different strains of S. typhimurium as target cells with the rat liver S-9 fraction and appropriate co-factors for metabolic activation of the chemical tested. The bacterial tester strains detect both mutations induced by base pair substitutions and intercalation (frame shift mutations). Usually 10(8)--10(9) cells of an overnight culture or an exponentially growing culture are incubated for 2-3 days with a mixture of S-9, co-factors, soft agar and the chemical on histidine-deficient agar. The S-9 fraction is obtained from the livers of rats pretreated with 500 mg/kg chlorinated biphenyls (Clophen A-50, Aroclor 1254) to obtain high metabolic activity. For reproducibility it is essential to standardize metabolic activity and protein content of the S-9 and to use three different concentrations thereof in the test system. Since solvents inhibit metabolic activation of the chemicals they must not exceed 4% of the final 2.6 ml incubate. Several independent studies have shown that between 85 and 93% of chemical carcinogens are mutagens in the test. Regarding extrapolation to man one has to consider that the test is preferentially adapted for metabolic activation of the chemicals, whereas inactivation processes are absent or are less active than in vivo. Thus, the test provides qualitative rather than quantitative information on mutagenic effects of a chemical.

Glutathione and glutathione S-transferases in the Salmonella mammalian-microsome mutagenicity test.

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH. The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione.

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538, and TA98 without metabolic activation. Presumably owing to conjugation with bacterial GHS, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.

Contact allergy as a therapeutic tool for alopecia areata: application of squaric acid dibutylester.

Squaric acid dibutylester (SADBE), a potent contact allergen, was tested for mutagenicity in the bacterial plate incorporation assay (Ames test), in the presence and absence of mammalian microsomes. In contrast to dinitrochlorobenzene which is mutagenic in this test, SADBE was found not to be mutagenic. In 53 patients with extensive or total alopecia areata, SADBE dissolved in acetone was applied weekly to one side of the head, the other side serving as control. In 46 patients (87%), hair regrew either exclusively on the treated side, or considerably faster and denser on this side. In some patients, continuous treatment failed to maintain the response. Persistent response was observed in 37 patients (70%). These results are essentially the same as those obtained with DNCB. Therefore, contact allergy is proposed as a therapeutic concept for alopecia areata.

Mutagenicity and chromosomal aberrations as an analytical tool for in vitro detection of mammalian enzyme-mediated formation of reactive metabolites.

1. Incubation of trichloroethylene, 1,1-dichloroethylene, vinylchloride, tetra-chlorocyclopentadiene, the nitroso derivatives of the pesticides Carbaryl, Prometryn, and Dodin in the presence of metabolically active mouse liver microsomes and bacteria as target cells were mutagenic, whereas tetrachloroethylene, 1,2 cis- and transdichloroethylene, hexachlorocyclopentadiene, carbontetrachloride, chloroform, halothane, trichlorofluoromethane and styrene were not activated to mutagenic species. 2. In a similar in vitro test system using freshly isolated human lymphocytes as target cells dimethylnitrosamine induced chromosomal aberrations. 3. It is concluded from the experiments that submammalian or mammalian in vitro cell systems with metabolically active liver microsomes are not only suitable to screen for chemical mutagens but to demonstrate formation of reactive intermediates, which are short lived and cannot be detected by chemical procedure.