Inger Florin

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by the metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and beta-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts.

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37 degrees C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0 degrees C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.

Cellular internalisation of Clostridium difficile toxin A

The cytopathogenic effect of toxin A from Clostridium difficile was studied in cultured human lung fibroblasts. The final effect was dependent on toxin concentration and exposure time. Binding of the toxin to cells occurred at 0 degrees C as well as at 37 degrees C. The latency before appearance of the cytopathogenic effect was dose-dependent with a minimum of 45 min. The appearance of a cytopathogenic effect in toxin-treated cells was prevented by the addition of trypsin, antitoxin, lysosomotropic agents, inhibitors of the energy metabolism, 200 mM KCl, 20 mM benzyl alcohol and by incubation at 18 degrees C. Several inhibitors of lysosomal proteases did not prevent the appearance of the cytopathogenic effect. When the extracellular pH was lowered to 4.5 for 5 min immediately after toxin binding the period of latency was significantly shortened. Likewise, the protective effects of lysosomotropic agents were abolished by lowering the extracellular pH. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were less sensitive to toxin A than wildtype cells. The results indicate that cellular internalisation of toxin A is necessary for intoxication. Moreover, we postulate that the toxin needs some sort of enzymatic activation which can take place only after exposure of the toxin to a low pH.

Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B

The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin. Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 degrees C and in the presence of 200 mM KCl or 1-20 mM benzyl alcohol, i.e. at conditions when the fusion between endosomes and lysosomes is prevented. Fibroblasts treated with toxin at 37 degrees C but transferred to 18 degrees C within 10 min were also completely protected, whereas transfer to 18 degrees C later during the latency resulted in only partial protection. KCl was also protective upon addition after the toxin binding step. Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive. The resistance was not overcome by applying a low extracellular pH. The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol. In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication.

Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B

The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied. ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1-5 mM range) delay in the appearance of the cytopathogenic effect. With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h. Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction. The protective effect of ATP did not depend on its energy producing ability. Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells. ATP was protective even upon addition 10 min after the toxin binding step. In the presence of ATP, the toxin remained accessible to neutralization with antitoxin. In analogy with the P-site on diphtheria toxin, we postulate that C. difficile toxin B contains a polyphosphate-binding site. This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step.

Mutagenicity of some indoles and related compounds in the Ames test.

A series of indole derivatives and some analogues were tested for mutagenic activity using a histidine-requiring mutant of Salmonella typhimurium (TA 98). Quantitative tests were performed with and without addition of a liver fraction (S-9) from rats induced with Aroclor 1254. Three compounds, 2,3-dimethylindole, 2,3,5-trimethylindole and 2,3,6-trimethylindole, showed a mutagenic effect which was not increased on metabolic activation. The influence on the mutagenic activity of the number and positions of the substituents attached to the indole skeleton is discussed.

Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B

A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B. The mutant, termed CdtR-Q, was 10(4) times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10(3) times more resistant). The resistance was overcome by increasing the dose of toxin. The resistance has been stable after cultivation for 40 generations in the absence of toxin. The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells. The plating efficiency was about half that of the wild-type, whereas the growth rate was the same. The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D. It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membrane-damaging toxins with different mechanisms of action, with one exception; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type. The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli. The results are consistent with a mutation affecting the microfilaments in the cytoskeleton.

ADP-ribosylation in cultured cells treated with Clostridiumdifficile toxin B

In cultured fibroblasts intoxicated with Clostridium difficile toxin B, a radioactive moiety was transferred from [14C-adenosine]NAD, but not from [14C-nicotinamide] NAD, into a cellular protein (MW 90,000). No labeling was detected in toxin-treated cultures not yet showing any toxin-induced cytopathogenic effect, whereas maximal labeling was obtained in cultures with about half of the cells showing a cytopathogenic effect. The radioactivity was removed from the substrate by treatment with snake venom phosphodiesterase. The results suggest that ADP-ribosylation of a cellular protein occurs in toxin B-treated cells and that this reaction may be responsible for development of the cytopathogenic effect.

ADP-RIBOSYLATION IN CLOSTRIDIUM DIFFICILE TOXIN-TREATED CELLS IS NOT RELATED TO CYTOPATHOGENICITY OF TOXIN B

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.

Dithiothreitol generates an activated 250,000 mol. wt form of Clostridium difficile toxin B

The potent cytotoxin of Clostridium difficile, toxin B, is internalized by endocytosis and activated intracellularly by an unidentified mechanism. Here it is shown that dithiothreitol treatment of toxin B resulted in (1) a mol. wt of 250,000 which is the smallest species of this toxin shown to be cytotoxic; (2) an increased endpoint titre; and (3) translocation of plasma membrane-bound toxin across the membrane at pH 4.5. Treatment with dithiothreitol can thus mimic intracellular activation of the toxin. Radiolabelling of highly purified toxin with retained activity, as well as the 32 N-terminal amino acids and the amino acid composition, is also presented.