Hematopoiesis toxicity induced by 4-methylumbelliferon determined in an invertebrate model organism.

Umbelliferone has potential value as it has an inhibitory effect on tumor cells; however, its impact on an animal's circulatory system and hematopoietic function has not been reported. In this study, 4-methylumbelliferon (4-MU), an umbelliferone derivative, was used as a model drug, and its potential toxicity on hemocytes and hematopoietic organs (HOs) was investigated using an invertebrate animal model, the silkworm, Bombyx mori. The results showed that the level of reactive oxygen species in HOs increased when larvae (third day of the fifth instar) were orally exposed to 4 mM 4-MU for 8 min, followed by the induction of improved antioxidative metabolism of coenzymes in hemolymph. Exposure to 4-MU also significantly upregulated the expression levels of several genes in the hemolymph and fat body (a detoxification tissue similar to the liver in mammals) including antimicrobial peptide gene cecropinA and moricin, and a phagocytosis-related gene, tetraspanin E, suggesting an increased antioxidant level and antimicrobial ability of the circulatory system. However, the percentage of dead hemocytes increased and hematopoiesis significantly decreased in HOs, indicating the toxic effect of 4-MU on hemocytes and hematopoiesis, despite it inducing enhanced antioxidant and antimicrobial activity in the circulatory system.

Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice.

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.

The lack of mutagenicity of aryl 4-guanidinobenzoates in the transplacental micronucleus assay in mice.

Two acrosin inhibitors, 4'-methylumbelliferyl 4-guanidinobenzoate and 2'-carbomethoxyphenyl 4-guanidinobenzoate, were tested for mutagenicity in the transplacental micronucleus assay and the mouse bone marrow micronucleus assay. The compounds were administered intraperitoneally at doses of 125 mg/kg and 250 mg/kg to pregnant mice. Fetal peripheral blood and maternal bone marrow cells were examined at 36 h for the frequency of micronucleated polychromatic erythrocytes. Neither compound induced micronuclei in maternal or fetal tissues. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was not affected by the drug treatments indicating that the compounds had no effect on the cell cycle or mitosis in these tissues and that they were not cytotoxic. Both compounds, which show promise as vaginal contraceptives, were not mutagenic in this study.

Antifertility activity of systemically administered proteinase (acrosin) inhibitors.

Low molecular weight, synthetic proteinase inhibitors that inhibit sperm-associated acrosin, were released systemically in female mice at a constant rate from minipumps. The release was timed so that, after mating, the minipumps were depleted of inhibitor before blastocyst implantation took place. Three of the inhibitors: 4-aminobenzamidine (ABD), 4-nitrophenyl-4-guanidino-benzoate (NPGB) and 4-methylumbelliferone-4-guanidinobenzoate (MUGB) caused a 50% decrease in fertility, the last two at very low concentrations. The fourth inhibitor, benzamidine (BD), which is also the weakest inhibitor of mouse acrosin and in vitro fertilization, had no effect. These results show that at least one of the processes leading to fertilization or early blastogenesis, is dependent on proteolytic activity and that the systemic application of proteinase inhibitors inhibits conception. MUGB possessed low toxicity and a high margin of safety, encouraging the development of phenol derivatives of guanidinobenzoic acid as contraceptive agents.

Development of the biochemical and morphological changes induced by administration of a beta-xyloside to chick embryos.

4-Methylumbelliferyl beta-D-xyloside was administered to 9-day-old chick embryos, and the morphological and chemical changes in the embryo were followed daily. Increases in wet weight, Na and Cl content, and visible edema were detectable at 10 days and fully apparent at 11 days. Dry weight increased to the same extent in control and treated embryos for four days, but then diverged. The degree of sulfation of chondroitin sulfate was slightly less in treated than control embryos at 10 days, and reached a steady low value at 11 days. Analysis of glycosaminoglycans in skin, muscle, and aorta showed an increase in chondroitin and its sulfates in the two former tissues but not the latter. In muscle and aorta, the degree of sulfation of chondroitin sulfate was markedly reduced; but in skin the results suggested a more complex picture in which the normal metabolism of glycosaminoglycans was altered. A possible physiological role is suggested for chondroitin sulfate in embryonic soft tissues.