Comparative study of the effects of 1,3,4-thiadiazolium mesoionic derivatives on energy-linked functions of rat liver mitochondria.

The main goal of this work was to investigate the relationship between the effects of three new 1,3,4-thiadiazolium mesoionic derivatives on mitochondrial bioenergetics and their previously described chemical structure and antimelanoma activity. The 4-phenyl-5-(2'-Y, 4'-X or 4'-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides differed from each other only in the cinnamoyl ring substituent: MI-J, X=OH; MI-F, X=F; MI-2,4diF X=Y=F. The state 3 respiratory rate was strongly decreased by all derivatives, reaching total inhibition of MI-4F and MI-2,4diF (130nmolmg(-1) protein), when glutamate plus malate were used as substrate. State 3 inhibition was less accentuated with succinate as substrate. Analyses of segments of the respiratory chain indicated complexes I and IV as sites inhibited by the derivatives. State 4 respiration was strongly stimulated by the three derivatives, and was characterized as an uncoupling effect, which was more intense for MI-4F. This stimulus was so pronounced that the values of RCC and ADP/O ratio were only calculated for the lowest concentration (6.5nmolmg(-1) protein). In intact mitochondria, the ATPase activity was increased dramatically by approximately 120%, approximately 207% and approximately 261% for MI-J, MI-4F and MI-2,4diF (32.5nmolmg(-1) protein), respectively. In conclusion, the presence of fluorine substituent in the cinnamoyl ring intensifies the effect of mesoionic compounds on mitochondrial functions and, in this context, hydrophobicity is more important than the electronic effect, which was correlated to antimelanoma activity described previously for these compounds.

The inhibition of lipoperoxidation by mesoionic compound MI-D: a relationship with its uncoupling effect and scavenging activity.

Important biological activities have been described for mesoionic compounds. We previously reported that MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride) inhibited the respiratory chain, collapsed the transmembrane potential, and stimulated ATPase activity in intact rat liver mitochondria. It is known that drugs that affect mitochondrial membrane potential may facilitate the induction of cell death by apoptosis. Mitochondria have also a central role in the generation of reactive oxygen species, therefore it would be important to investigate how MI-D could affect processes related to oxidative stress. In this work, we evaluated the effects of MI-D on the lipoperoxidation and its ability to scavenge free radicals. Interestingly, it was observed that MI-D promoted a strong inhibition of the lipoperoxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria (95%+/-0.27 at the highest concentration of 80 nmol mg(-1) protein) in a dose-dependent manner. However, at the same concentration its effect was less intense (22%+/-3.46) when the lipoperoxidation was initiated by peroxyl radicals generated from the azocompound AAPH. Lipid peroxidation in both coupled and uncoupled submitochondrial particles initiated with Fe(2+)/NADH was also inhibited by MI-D. The inhibition was about four times greater in coupled particles (approximately 34% at 80 nmol mg(-1) protein) in relation to uncoupled. MI-D inhibited the soybean phosphatidylcholine liposomes lipoperoxidation in a dose-dependent manner (5-80 microM) regardless of the radical being generated in lipid or aqueous phase. The mesoionic compound showed ability of scavenging superoxide radical (7, 11 and 31% for 25, 38 and 80 microM, respectively). Our results strongly suggest that the inhibition of lipoperoxidation promoted by MI-D is due to its scavenger action and to its previously described uncoupling effect.

Production of cachexia mediators by Walker 256 cells from ascitic tumors.

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 +/- 1.56 and 4.05 +/- 1.99 nmol NO per 10(7) cells, respectively). When isolated from a 6-day-old tumor, a significantly lower production of IL-6 and higher production of TNF-alpha than in cells from a 4-day-old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE(2) by Walker 256 cells isolated from the 6-day-old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time.

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria.

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.

Biological evaluation of some selected cyclic imides: mitochondrial effects and in vitro cytotoxicity.

Cyclic imides such as succinimides, maleimides, glutarimides, phthalimides and their derivatives contain an imide ring and a general structure -CO-N(R)-CO- that confers hydrophobicity and neutral characteristic. A diversity of biological activities and pharmaceutical uses have been attributed to them, such as antibacterial, antifungal, antinociceptive, anticonvulsant, antitumor. In spite of these activities, much of their action mechanisms at molecular and cellular levels remain to be elucidated. We now show the effects of several related cyclic imides: maleimides (S2, S2.1, S2.2, S3), glutarimides (S4, S5, S6), 4-aminoantipyrine derivatives (L1, F1, AL1, F1.14, F1.2) and sulfonated succinimides (RO1, FA, FE, FD, MC, DMC) on isolated rat liver mitochondria, B16-F10 melanoma cell line, peritoneal macrophages and different bacterial streams. The effects on mitochondrial respiratory parameters, cell viability and antibacterial activity were also evaluated. The results indicated that S3, S5 and S6 caused an increased oxygen consumption in the presence of ADP (state III) or its absence (state IV), while all other compounds decreased those parameters at different degrees of inhibition. All the compounds decreased the respiratory control coefficient (RCC). Loss of cell viability of peritoneal macrophages and the B16-F10 cell line was observed, L1 and S2.1 being more effective. S1, S2, S3, L1 and F1 compounds showed antibacterial activity at experimental concentrations.

Effects of citrinin on iron-redox cycle.

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.

Interference of MI-D, a new mesoionic compound, on artificial and native membranes.

MI-D (4-phenyl-5-(4-nitrocinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride), a new mesoionic compound, decreased the rate of swelling induced by valinomycin-K+, as well as induced swelling in the presence of nigericin-K+. Shrinkage was also affected, suggesting interference with the inner mitochondrial membrane, which would affect both fluidity and elasticity. Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions respectively, of the DMPC and native membranes, indicated that MI-D shifts the midpoint of phase transition to higher values and orders of the fluid phase. These alterations in membrane fluidity are thus related to MI-D effects on the energy-linked functions of mitochondria.