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Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro.It was found that a synthetic P-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity.The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-α induction and NF-κB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-α release and activated NF-κB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-α and NF-κB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-α induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-α release and NF-κB activation in THP-1 cells.

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UV stabilizers used in food contact plastics were tested for their estrogenic activity by the yeast two-hybrid assay. Among 11 kinds of UV stabilizers, 2-hydroxy-4-methoxybenzophenone and 2,2′-dihydroxy-4-methoxybenzophenone displayed estrogenic activity, while salicylate, benzoate and benzotriazole derivatives and a benzophenone derivative had no activity. Therefore, benzophenone and 19 kinds of hydroxylated derivatives were further studied. Of these, 15 chemicals showed estrogenic activity. The strongest activity was by 2,4-dihydroxybenzophenone, 4hydroxy-4′-chlorobenzophenone, 4-hydroxybenzophenone and 2,3,4-trihydroxybenzophenone. Their activities were stronger than that of bisphenol A which was recognized as a potential endocrine disruptor. The following structureactivity relationships of benzophenones were obtained. The activity of the benzophenones with a hydroxyl group at the 3 or 4-position was positive and rather strong, though that of other benzophenones without a hydroxyl group at the 3 or 4-position was negative or weakly positive. The effect of the hydroxyl group in the phenol moiety were in order of 4-> 3->> 2-position. A hydroxyl group added at the 2-position of the 4-hydroxylated benzene ring enhanced the activity. On the other hand, a hydroxyl group added to the benzene ring of the hydrophobic moiety reduced the binding, while the chloro group enhanced it. Some of these relationships might possibly hold for other estrogenic chemicals that possess two benzene rings.

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