Maria T. Runnegar

NAMING OF CYCLIC HEPTAPEPTIDE TOXINS OF CYANOBACTERIA (BLUE-GREEN-ALGAE)

UNIV ILLINOIS,COLL VET MED,URBANA,IL 61801; USA,MED RES INST INFECT DIS,DIV PATHOPHYSIOL,FREDERICK,MD 21701; UNIV NEW ENGLAND,DEPT BIOCHEM MICROBIOL & NUTR,ARMIDALE,NSW 2351,AUSTRALIA; UNIV ALBERTA,DEPT BOT,EDMONTON T6G 2E1,ALBERTA,CANADA; MEIJO UNIV,FAC PHARM,TEMPA KU,NAGOYA,AICHI 468,JAPAN; CHEM RES & DEV CTR,ABERDEEN,MD 21701; NATL BOT GARDENS,CLAREMENT,SOUTH AFRICA; ACAD SINICA,INST HYDROBIOL,WUHAN,PEOPLES R CHINA; NORWEGIAN INST WATER RES,OSLO 3,NORWAY; UNIV HAWAII MANOA,DEPT CHEM,HONOLULU,HI 96822; UNIV ILLINOIS,SCH CHEM SCI,URBANA,IL 61801; UNIV CALIF LOS ANGELES,DEPT MICROBIOL,LOS ANGELES,CA 90024; TOKYO METROPOLITAN RES LAB PUBL HLTH,SHINJUKU KU,TOKYO 160,JAPAN

Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes

Cylindrospermopsin (CY) is a naturally occurring alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in humans. We previously showed that CY is cytotoxic to primary cultures of rat hepatocytes and that CY lowers cell reduced glutathione (GSH) at nontoxic doses. Lower cell GSH also potentiates CY-induced cytotoxicity (Runnegar et al., Biochem Biophys Res Commun 201: 235-241, 1994). Our current work examined the mechanism of the fall in cell GSH induced by CY. We excluded several possible explanations for the loss in GSH, namely increased formation of oxidized glutathione (GSSG), increased GSH efflux, hidden forms of GSH, decreased GSH precursor availability, or decreased cellular ATP level. To address whether the fall in GSH was due to decreased GSH synthesis or increased GSH consumption, we examined the rate of fall in total GSH after 5 mM buthionine sulfoximine (BSO, an irreversible inhibitor of GSH synthesis) treatment. The rates of fall in total GSH (nmol/10(6) cells/hr) were 8.2 +/- 2.5, 6.0 +/- 1.7 and 5.9 +/- 1.3 for control, 2.5 microM and 5 microM CY-pretreated cells, respectively. This suggests that the fall in GSH induced by CY was due to the inhibition of GSH synthesis rather than increased consumption, because in the latter case the rate of fall in GSH would have been accelerated by CY pretreatment. Furthermore, excess GSH precursor (20 mM N-acetylcysteine), which supported GSH synthesis in control cells, did not prevent the fall in GSH or toxicity induced by CY. Treatment of cells with the cytochrome P450 inhibitor alpha-naphthoflavone protected partially from CY-mediated toxicity and from the fall in cell GSH. Thus, it is likely that cytochrome P450 is involved in the metabolism of CY, and the metabolite(s) that is generated may be more toxic and/or potent in inhibiting GSH synthesis. Inhibition of GSH synthesis is most likely an important factor in the cytotoxicity of CY.

S-adenosylmethionine and its metabolite induce apoptosis in HepG2 cells: Role of protein phosphatase 1 and Bcl-xS

S‐adenosylmethionine (SAMe) and its metabolite 5′‐methylthioadenosine (MTA) are proapoptotic in HepG2 cells. In microarray studies, we found SAMe treatment induced Bcl‐x expression. Bcl‐x is alternatively spliced to produce two distinct mRNAs and proteins, Bcl‐xL and Bcl‐xS. Bcl‐xL is antiapoptotic, while Bcl‐xS is proapoptotic. In this study we showed that SAMe and MTA selectively induced Bcl‐xS in a time‐ and dose‐dependent manner in HepG2 cells. There are three transcription start sites in the human Bcl‐x gene which yield only Bcl‐xL in control HepG2 cells. SAMe and MTA treatment did not affect promoter usage, but while one promoter yielded only Bcl‐xL, the other two yielded both Bcl‐xL and Bcl‐xS, with Bcl‐xS as the predominant messenger RNA (mRNA) species. Trichostatin A, 3‐deaza‐adenosine, cycloleucine, and okadaic acid had no effect on Bcl‐xS induction by SAMe or MTA. Calyculin A and tautomycin, on the other hand, blocked SAMe and MTA‐mediated Bcl‐xS induction and apoptosis in a dose‐dependent manner. SAMe and MTA increased protein phosphatase 1 (PP1) catalytic subunit mRNA and protein levels and dephosphorylation of serine–arginine proteins, with the latter blocked by calyculin A. The effects of SAMe and MTA on Bcl‐xS, PP1 expression, and apoptosis were also seen in 293 cells, but not in primary hepatocytes. Induction of Bcl‐xS by ceramide in HepG2 cells also resulted in apoptosis. In conclusion, we have uncovered a highly novel action of SAMe and MTA, namely the ability to affect the cellular phosphorylation state and alternative splicing of genes, in this case resulting in the induction of Bcl‐xS leading to apoptosis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:221–231.)

Inhibition of nuclear protein phosphatase activity in mouse hepatocytes by the cyanobacterial toxin microcystin-LR

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin and protein phosphatase inhibitor that contaminates water reservoirs worldwide. MCLR localizes to the cytosol of hepatocytes, however, immunohistochemical studies indicate that it accumulates in the nucleus. MCLR toxicosis is associated with decreased hepatic protein phosphatase activity, but effects in nuclear protein phosphatase activity have not been investigated. Balb/c mice were given lethal (100 microg/kg) or sublethal (12, 23 and 45 microg/kg) i.p. doses of MCLR and hepatic nuclear extracts were analyzed for protein phosphatase 1 and 2A activity. There was profound inhibition of nuclear protein phosphatase activity within 50 min of lethal dosing, however an inhibition was not detected with sublethal doses. MCLR immunohistochemistry revealed widespread lobular staining in the lethal group and centrilobular staining in the sublethal groups. At the cellular level there was nuclear and cytoplasmic staining of equal intensity. As an indicator of nuclear protein phosphatase activity, the phosphorylation of p53, a nuclear phosphoprotein and known substrate for protein phosphatases 1 and 2A, was evaluated. Balb/c mice were treated with sublethal doses of MCLR or saline vehicle after induction of hepatic p53 by the DNA damaging agent diethylnitrosamine (DEN). P53 was immunoprecipitated and probed with phosphoserine specific antibodies by Western blotting. There was greater phosphoserine reactivity of p53 protein in animals treated with MCLR relative to saline treated controls, consistent with increased phosphorylation of serine sites. It is concluded that an interaction of this toxin with nuclear protein phosphatases occurs within 50 min of lethal dosing, which leads to a profound inhibition of enzymatic activity. Even sublethal doses of MCLR that do not result in significant inhibition of activity in bulk nuclei, result in detectable changes in phosphorylation of p53.