Toxicity evaluation of new antifouling compounds using suspension-cultured fish cells.

A simple, rapid toxicity test was developed using the suspension-cultured fish cell line CHSE-sp derived from chinook salmon Oncorhynchus tshawytscha embryos in order to assess the toxicity of new marine antifouling compounds. The compounds tested were copper pyrithione, Diuron, Irgarol 1051, KH101, Sea-Nine 211, and zinc pyrithione, all of which have been nominated in Japan as possible replacements for organotin compounds. The in vitro acute toxicity (24-h EC50) of the six compounds to these fish cells was evaluated using the dye Alamar Blue to determine cell viability, and then correlated with the results of in vivo chronic toxicities (28-day LC50) to juvenile rainbow trout Oncorhynchus mykiss. The suspension-cultured fish cells were found to be suitable for the screening of such chemicals before performing an in vivo test. The toxicities of the test compounds obtained from both tests, shown in decreasing order, were as follows: copper pyrithione > zinc pyrithione > KH101 > or = Sea-Nine 211 > Diuron > Irgarol 1051. The herbicides Diuron and Irgarol 1051 showed the least toxicity, while the pyrithiones had the greatest toxicity.

Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.

Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells.

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA >> (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.

Inhibitory effect of a new antibiotic, guanine 7-N-oxide, on the replication of several RNA viruses.

Guanine 7-N-oxide (G-7-Ox) was examined for its antiviral activity against 9 viruses based on plaque reduction, neuraminidase activity reduction, a fluorescent antibody technique or ELISA. The following viruses were included in the tests: influenza, Sendai, simian virus 5 (SV5), respiratory syncytial, western equine encephalitis, Japanese encephalitis, vesicular stomatitis, rabies and polio. G-7-Ox showed broad anti-RNA viral activity against all viruses tested, except for poliovirus. Inhibition of persistent SV5 infection by G-7-Ox indicates that its antiviral activity is independent of cytotoxicity.

2-Methylpropyl ester of 3-(adenin-9-yl)-2-hydroxypropanoic acid. Mechanism of antiviral action in vaccinia virus-infected L929 cells.

Alkyl esters of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid (AHPA) were shown recently to be broad spectrum antiviral agents (De Clercq E and Holy A, J Med Chem 28: 282-287, 1985). It was postulated that these alkyl esters function as prodrugs by undergoing hydrolysis catalyzed by cellular esters to AHPA, a known inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. In this study, we describe the metabolic fate of the 2-methylpropyl ester of AHPA (AHPA-iBu) in murine L929 cells. When AHPA-iBu was included in the culture medium, it was taken up rapidly by murine L929 cells. The uptake was time- and concentration-dependent, resulting in the intracellular accumulation of the free acid, AHPA. Treatment with AHPA-iBu caused inhibition of cellular AdoHcy hydrolase in both a time- and a dose-dependent manner. Complete inhibition of the enzyme was achieved after a 1-hr incubation in culture medium containing 50 microM AHPA-iBu. The inhibition of the enzyme caused cellular accumulation of AdoHcy and a significant increase in the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet). Partial recovery of the AdoHcy hydrolase activity in L929 cells treated with 50 microM AHPA-iBu was observed after 24 hr. This recovery of enzyme activity was paralleled by a significant decrease in the cellular levels of AdoHcy and the ratio of AdoHcy/AdoMet. AHPA-iBu also exerted an inhibition (IC50 = 0.17 microM) of vaccinia virus plaque formation in monolayers of L929 cells. A 1 microM concentration of AHPA-iBu, which caused 80% inhibition of plaque formation, produced a 17-fold increase in AdoHcy content in drug-treated, virus-infected cells versus non-drug-treated, virus-infected cells and a 15% undermethylation of the poly(A)+ RNA. These data show that AHPA-iBu is a prodrug for AHPA which inhibits cellular AdoHcy hydrolase. The inhibition of this enzyme elevates cellular levels of AdoHcy, creating an unfavorable environment which suppresses replication of vaccinia virus.

Mechanism of the synergistic antiviral and cytostatic activity of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid isobutyl ester and D,L-homocysteine.

In a previous report (De Clercq E, Cools M and Balzarini J, Biochem Pharmacol 38: 1771-1778, 1989) we showed that homocysteine (Hcy) enhanced the antiviral and cytostatic activity of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors. The mechanism of synergistic action between Hcy and the isobutyl ester of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(RS)-AHPA] has been the subject of the present study. The selectivity index of (RS)-AHPA against vaccinia virus in murine L929 cells was significantly increased if the drug was combined with 1 or 3 mM Hcy. Even if Hcy was added as late as 12 hr after (RS)-AHPA, a synergistic antiviral activity was noted. Treatment of the L929 cells with (RS)-AHPA caused a significant increase in AdoHcy levels, and these levels were further increased if, in addition to (RS)-AHPA, Hcy (1 mM) was added to the cell cultures. Double-pulse label experiments showed that the additional AdoHcy built up after the combined treatment of (RS)-AHPA with Hcy did not originate from S-adenosylmethionine (via transmethylation reactions), but resulted from residual AdoHcy hydrolase activity (in the synthetic direction). To maintain sufficient levels of AdoHcy, AdoHcy hydrolase activity must be inhibited in the hydrolytic direction.

Elucidation of the mechanism by which homocysteine potentiates the anti-vaccinia virus effects of the S-adenosylhomocysteine hydrolase inhibitor 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine.

9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine (DHC), a specific inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase, has been used in this study to elucidate the mechanism by which DL-homocysteine (Hcy) potentiates the antiviral effects of AdoHcy hydrolase inhibitors as reported by De Clercq [Biochem. Pharmacol. 36:2567-2575 (1987)]. The potentiating effects of Hcy on the antiviral effects of DHCA were determined using murine L929 cells infected with vaccinia virus. When virus-infected cells were incubated with DHCA alone or in combination with various concentrations of Hcy, the following IC50 values (concentrations of the drug required to reduce by 50% viral plaque formation) were observed: 0.30 microM (0 mM Hcy), 0.15 microM (0.3 mM Hcy), 0.09 microM (1.0 mM Hcy), and 0.04 microM (3.0 mM Hcy). In the drug combination studies, increased cellular toxicity, compared with DHCA alone, was observed only at the highest concentration of Hcy (3.0 mM); thus, at lower concentrations Hcy increased the antiviral effectiveness [ID50 (concentration of the drug required to reduce the increase in cell number by 50%)/IC50] of DHCA. For example the following ID50/IC50 values were observed for DHCA alone or in combination with Hcy: 64 (0 mM Hcy), 113 (0.3 mM Hcy), 151 (1.0 mM Hcy), and 88 (3.0 mM Hcy). In these studies, Hcy was also observed to potentiate the increase in cellular levels of AdoHcy and the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet) in DHCA-treated cells. In earlier studies, our laboratory has shown that antiviral effects of DHCA are caused by only slight elevations in intracellular levels of AdoHcy [from 50 pmol/mg of protein (controls) to 100-200 pmol/mg of protein (drug-treated)] and slight elevations in the ratios of AdoHcy/AdoMet [from 0.05-0.1 (control) to 0.15-0.20 (drug-treated)]. Thus, in the presence of Hcy, lower concentrations of DHCA are needed to increase the intracellular concentration of AdoHcy and the AdoHcy/AdoMet ratio to levels that suppress replication of vaccinia virus. Murine L929 cells were shown to contain DHCA-sensitive and DHCA-insensitive forms of AdoHcy hydrolase. Based on the results of labeling experiments using [2,8-3H]adenosine and [35S]methionine, the elevated levels of AdoHcy were shown to arise from the reaction of [2,8-3H]adenosine and Hcy, catalyzed by the DHCA-insensitive form of AdoHcy hydrolase.

Relationship between intracellular concentration of S-adenosylhomocysteine and inhibition of vaccinia virus replication and inhibition of murine L-929 cell growth.

9-(trans-2',trans-3'-Dihydroxycyclopent-4'-enyl)-adenine (compound 1) and -3-deazaadenine (compound 2), which are specific inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were reported earlier by our laboratory (M. Hasobe, J. G. McKee, D. R. Borcherding, and R. T. Borchardt, Antimicrob. Agents Chemother. 31:1849-1851, 1987) to have anti-vaccinia virus activity with reduced murine L-929 cell toxicity compared with the prototype compound neplanocin A. In this study, we showed that the antiviral and cytotoxic effects of compounds 1 and 2 can be related to intracellular concentrations of AdoHey, which are elevated in cells treated with these inhibitors of AdoHcy hydrolase. For example, concentrations of analogs 1 and 2 that produce 50% inhibition of vaccinia virus replication caused only slight elevations in intracellular levels of AdoHcy (from 50 [controls] to 100 to 125 [drug-treated cells] pmol/mg of protein) and elevations in the ratios of AdoHcy/S-adenosylmethionine (from 0.05 to 0.1 [controls] to 0.15 to 0.19 [drug-treated cells]). In contrast to the extreme susceptibility of virus replication to slight elevations in intracellular AdoHcy, cell viability was quite tolerant to higher levels of this metabolite. For example, concentrations of analogs 1 and 2 that produced 50% inhibition of L-929 cell replication caused significant increases in intracellular levels of AdoHcy (to 825 to 950 pmol/mg of protein) and elevations in AdoHcy/S-adenosylmethionine ratios (approximately 1.3). These data make it possible to assign a therapeutic index of 7 to 8 to these compounds on the basis of the comparison of intracellular levels of AdoHcy that caused 50% inhibition of vaccinia virus replication with those that caused 50% inhibition of L-929 cell replication.

Antiviral activities and the mechanism of 9-beta-D-ribofuranosyl-6-alkylthiopurines on several RNA viruses from animals.

A series of 9-beta-D-ribofuranosyl-6-alkylthiopurines (6-alkyl TI) were found to inhibit in vitro replication of infectious hematopoietic necrosis virus (IHNV), human influenza virus (IFV) and respiratory syncytial virus (RSV) with IC50 values of about 0.06 microgram/ml, 0.7-1.5 micrograms/ml and 1-3 micrograms/ml, respectively. Viral RNA synthesis in infected cells in the presence of actinomycin D was inhibited by treatment with the compounds dose-dependently. It was also found that the decrease of rNTP pool size in infected cells was remarkably dose-dependent. From these findings, the mode of antiviral action of these compounds may be explained by rNTP imbalance in the treated group.

Inhibitory effect of sulphur-containing purine nucleoside analogues on replication of RNA viruses: selective antiviral activity against influenza viruses.

Four sulphur-containing purine nucleoside analogues: 6MP, 6-thioinosine, 6-methylthioinosine and 6-ethylthioinosine, were examined for antiviral activity against some RNA viruses. All compounds extensively inhibited the replication of influenza viruses but had no inhibitory effect on other RNA viruses: Sendai, RS, vesicular stomatitis and western equine encephalitis viruses.

Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 11. Molecular dissections of neplanocin A as potential inhibitors of S-adenosylhomocysteine hydrolase.

A series of 9-(hydroxyalkenyl)purines (adenines and 3-deazaadenines), which are analogues of neplanocin A, were synthesized. The analogues were tested as inhibitors of bovine liver and murine L929 cell S-adenosyhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) and as inhibitors of vaccinia virus replication in murine L929 cells. Compounds 1b, 2a, 2b, 4a, 4b, 7, 9a, and 9b showed the best inhibitory effects toward bovine liver AdoHcy hydrolase, with compound 4b being the most potent. The compounds that were shown to be the most potent inhibitors of the bovine liver AdoHcy hydrolase all contained an allylic hydroxyl group in the cis position to the adenine or the 3-deazaadenine rings. It was concluded that the cis arrangement of the allylic hydroxyl groups in these acyclic compounds represented the minimum structural requirement of the trihydroxycyclopentenyl ring of neplanocin A to show inhibitory effects against AdoHcy hydrolase. The antiviral effects of these acyclic analogues were significantly less than neplanocin A; however, there appears to be a correlation between the antiviral activity and the inhibition of AdoHcy hydrolase for compounds 2a, 2b, 4a, 4b, and 7. Analogue 4b, which exhibited the best antiviral activity (IC50 = 70 microM) in this acyclic series, is substantially less potent than neplanocin A (IC50 = 0.08 microM) as an antiviral agent.

Effects of 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine and -3-deazaadenine on the metabolism of S-adenosylhomocysteine in mouse L929 cells.

Neplanocin A [(-)-9-[trans-2',trans-3'-dihydroxy-4'-(hydroxymethyl)-cyclopent-4 '- enyl]-adenine] and 9-[trans-2',trans-3'-dihydroxycyclopent-4'-enyl]-adenine (1) and -3-deazaadenine (2) are potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) in mouse L929 cells. When cells were treated for 15 min with varying concentrations of the drugs, the IC95 values (concentration needed to produce 95% inhibition of AdoHcy hydrolase) for neplanocin A, 1, and 2 were determined to be 0.2 microM, 0.5 microM, and 0.5 microM, respectively. Incubation of L929 cells with 1.0 microM concentrations of neplanocin A, 1, or 2 produced rapid inactivation of AdoHcy hydrolase (within 30 min the enzyme was 95% inhibited), which persisted for at least 72 hr. At lower concentrations (0.032 microM), substantial recovery of AdoHcy hydrolase activity was noted after 48 and 72 hr in cultures treated with neplanocin A but not in cultures treated with 1 or 2. L929 cells treated with neplanocin A, 1 or 2 showed a rapid increase in intracellular levels of AdoHcy (as well as the ratio of AdoHcy/S-adenosylmethionine). Cells treated with neplanocin A also contained significant amounts of S-neplanocylmethionine, whereas cells treated with 1 or 2 showed no evidence of the formation of a similar metabolite. When neplanocin A and adenosine were incubated in cell lysates, rapid conversion to neplanocin D and inosine, respectively, were observed, illustrating the affinity of these nucleosides for cellular adenosine deaminase. In contrast, when 1 and 2 were incubated in cell lysates, no evidence for deamination was observed. These data illustrate that compounds 1 and 2 retain the inhibitory activity of neplanocin A toward cellular AdoHcy hydrolase, producing elevated cellular levels of AdoHcy. However, by removing the 4'-hydroxymethyl group from neplanocin A, analogs 1 and 2 are no longer substrates for adenosine deaminase and adenosine kinase.

9-(trans-2',trans-3'-Dihydroxycyclopent-4'-enyl)-adenine and -3-deazaadenine: analogs of neplanocin A which retain potent antiviral activity but exhibit reduced cytotoxicity.

Two synthetic analogs of neplanocin A, which were shown in a separate study to be inhibitors of S-adenosylhomocysteine hydrolase and devoid of substrate activity with adenosine kinase, were found in this study to inhibit vaccinia virus replication in murine L929 cells but to have reduced cytotoxicity compared with that of the parent compound. These results confirm that S-adenosylhomocysteine hydrolase is the molecular target which mediates the antiviral effects of neplanocin A and that transformation by cellular adenosine kinase mediates its cytotoxic properties.

The synergism of nucleoside antibiotics combined with guanine 7-N-oxide against a rhabdovirus, infectious hematopoietic necrosis virus (IHNV).

Guanine 7-N-oxide was shown to have synergistic activity in combination with neplanocin A against a rhabdovirus, infectious hematopoietic necrosis virus (IHNV), as reported previously. We examined further the antiviral activity of guanine 7-N-oxide in combination with other nucleoside antibiotics against IHNV. Synergism was seen between guanine 7-N-oxide and D-eritadenine or cordycepin. It is considered that compounds inhibiting RNA methylation show synergism with guanine 7-N-oxide.

Antiviral activity and its mechanism of guanine 7-N-oxide on DNA and RNA viruses derived from salmonid.

Guanine 7-N-oxide produced by Streptomyces sp. was found to inhibit in vitro the replication of herpes virus (Oncorhynchus masou virus, OMV), rhabdo virus (infectious hematopoietic necrosis virus, IHNV) and a bi-segmented double-strand virus (infectious pancreatic necrosis virus, IPNV) derived from salmonids with IC50 values of about 10 micrograms/ml, 20 micrograms/ml and 32 micrograms/ml, respectively. The agent was not toxic for the host cells (chinook salmon embryo, CHSE-214) at the IC50 concentrations. Labeling of IHNV viral RNA and host cellular DNA and RNA with [3H]uridine and [3H]thymidine during drug treatment showed that guanine 7-N-oxide did not reduce the incorporation of these precusors into RNA and DNA. The anti-IHNV activity of guanine 7-N-oxide was enhanced synergistically by neplanocin A, an inhibitor of RNA methylation. The mechanism of action of guanine 7-N-oxide is discussed, in regard to maturation of viral messenger RNA including capping.