Intravenous injection of lipopolysaccharide and D-galactosamine, at doses of 0.2 micrograms/kg and 800 mg/kg, respectively, elicited massive hepatic necrosis within 24 hr in C3H/HeN mice. The plasma L-alanine aminotransferase (ALT, E.C. 2.6.1.2) or L-aspartate aminotransferase (AST, E.C. 2.6.1.1) activities at this point reached more than 2,000 IU/L. However, overt hepatic injury as evaluated by the plasma aminotransferase activities did not develop in mice in which only lipopolysaccharide or only D-galactosamine was injected. No tumor necrosis factor-like activities could be detected in the plasma of galactosamine- and lipopolysaccharide-injected mice as determined by the assay of cytotoxicity to highly tumor necrosis factor-sensitive L-P3 cells through the experimental period of 24 hr. However, passive immunization against mouse tumor necrosis factor-alpha with polyvalent rabbit anti-mouse tumor necrosis factor-alpha antiserum, which was able to neutralize the cytotoxic effects of recombinant mouse tumor necrosis factor-alpha on L-P3 cells, could protect the mice from the development of hepatic injury in a dose-dependent manner. Simultaneous injection of recombinant human tumor necrosis factor-alpha, instead of lipopolysaccharide, with 800 mg/kg of D-galactosamine in lipopolysaccharide-resistant C3H/HeJ mice sensitized the animals more than one thousand-fold to the development of hepatic injury. The livers appeared to be morphologically similar to those of galactosamine- and lipopolysaccharide-injected C3H/HeN mice.

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Mouse lymphoma L1210 cells require some thiol compounds (such as 2-mercaptoethanol) or feeder layer cells for their growth in normal culture media in vitro. We found that feeder layer cells (human diploid fibroblast IMR-90) constantly produce thiol compounds; cysteine was the major thiol compound accumulating in the culture medium. In the culture medium of L1210 cells, added cysteine was rapidly oxidized and was toxic to the cells at high concentrations. However, cysteine promoted growth of L1210 cells when it was added repeatedly to the medium at low concentrations. These results show that the major role of feeder layer cells is to provide cysteine continuously. The glutathione content of L1210 cells depended largely on the cysteine concentration in the medium. In normal culture media containing cystine but not cysteine, the cellular glutathione content decreased notably within a few hours. Cysteine had to be supplied repeatedly to keep the content high. Cystine promoted the cellular glutathione content at unphysiologically high concentrations. These results were attributable to the extremely low uptake rate of cystine by the cells as compared with that of cysteine. In the presence of 2-mercaptoethanol, the uptake of radioactive cystine by the cells was increased and a high cellular glutathione level was maintained. A thiol-independent variant of L1210 took up cystine far more rapidly than L1210. From these results we concluded that the deficiency of the cystine uptake limits the growth of L1210 cells in normal culture media.

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A new series of heterocyclic ring-containing benzoic acids was prepared, and the binding affinity and antagonism of its members against all-trans-retinoic acid were evaluated by in vitro assay systems using human promyelocytic leukemia (HL-60) cells. Structure-activity relationships indicated that both an N-substituted pyrrole or pyrazole (1-position) and a hydrophobic region, with these linked by a ring system, were indispensable for effective antagonism. Among the compounds evaluated, optimal antagonism was exhibited by 4-[4,5,7,8,9,10-hexahydro-7,7,10,-10-tetramethyl-1-(3- pyridylmethyl)anthra[1,2-b]pyrrol-3-yl]benzoic acid (31), 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)-5- thiaanthral[1,2-b]pyrrol-3-yl]benzoic acid (40), and 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3- pyridylmethyl)anthra[2,1-d]pyrazol-3-yl]benzoic acid (55), all of which possess a 3-pyridylmethyl group at the five-membered ring nitrogen atom.