Tetsuya Ohta

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR

SummaryCertain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the twol-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathioneS-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Structure‐Function Relationships of Microcystins, Liver Tumor Promoters, in Interaction with Protein Phosphatase

Microcystins, isolated from toxic blue‐green algae, are potent inhibitors of protein phosphatases 1 and 2A. Recently, we have reported that mierocystin LR has a potent tumor‐promoting activity on rat liver initiated with diethylnitrosamine. The structure of microcystins is unique in having an unusual ammo acid, 3‐amino‐9‐methoxy‐10‐phenyl‐2,6,8‐trimethyl‐deca‐4(E),6(E)‐dienoic acid (Adda), which is thought to be significant for the activity. Geometrical isomers at C‐7 in the Adda portion of microcystins, 6(Z)‐Adda microcystins LR and RR, have been isolated from cyanohacteria. To estimate their tumor‐promoting activities and to understand the importance of the Adda portion for activity, the maternal microcystins LR and RR and their isomers were subjected to examination of their interaction with protein phosphatases 1 and 2A and the release of glutamic pyruvic transaminase from rat liver. 6(Z)‐Adda microcystins LR and RR bound to protein phosphatases 1 and 2A, inhibited their activities and released glutamic pyruvic transaminase from rat liver into serum, ten to one hundred times more weakly than the maternal microcystins LR and RR. These results indicated that the conjugated diene with 4(E),6(E) geometry in the Adda portion is important in the interaction with protein phosphatases.

Two significant aspects of microcystin-LR: specific binding and liver specificity

Microcystin-LR is a unique and potent liver tumor promoter, belonging to the okadaic acid class compounds. Although microcystin-LR is a potent inhibitor of protein phosphatases 1 and 2A, as is okadaic acid, microcystin-LR has liver specificity dominance. Two significant aspects, specific binding and liver specificity of [3H]dihydromicrocystin-LR, a reduced form of microcystin-LR, were studied and compared with those of [3H]okadaic acid. [3H]-Dihydromicrocystin-LR had higher affinity for the receptors in both the particulate and cytosolic fractions of rat liver and various tissues than had [3H]okadaic acid. Intraperitoneal administration of [3H]dihydromicrocystin-LR into mice resulted in the highest uptake into the liver, 71.5 +/- 6.9% of the total administered radioactivity, whereas p.o. administration resulted in less than 1% uptake into the liver, suggesting that the mechanisms of the incorporation of microcystin-LR into the liver by i.p. and p.o. administrations are greatly different. The presence of associated forms of [3H]dihydromicrocystin-LR with macromolecules in the liver indicates a need for further investigation.

Expression of the tumor necrosis factor alpha gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes.

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor α gene (TNFα) and early-response genes in rat liver after its i.p. administration, and since TNFα had tumor-promoting activity in vitro, it is possible that TNFα itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of theTNFα gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly inducedTNFα gene expression in rat hepatocytes, as well as TNFα release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of theTNFα gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 μM nodularin or microcystin-LR induced expression of the c-jun, jun B,jun D, c-fos, fos B andfra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that theTNFα gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.

Limited diffusibility of gene products directed by a single nucleus in the cytoplasm of multinucleated myofibres

Two types of β‐galactosidase genes, whose products are distributed in the nucleus (Nα‐gal) or cytoplasm (Cβ‐gal), were injected with fructose intramuscularly into the quadriceps of adult mice. Regionally restricted and overlapped distributions of both gene products were observed in the myofibres. These findings indicate that Nβ‐gal is incorporated into the nucleus responsible for its synthesis and that Cβ‐gal becomes located in the vicinity of the nucleus after its synthesis. This restricted location of C β‐gal in myofibres remained unchanged during the development of infant mouse muscle. Thus, the gene products directed by the nucleus of myofibres seem to show limited diffusibility, suggesting a universal localization of subcellular domains in myofibres.