Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters K(m) (19.5 +/- 4.5 microM) and V(max) [1.5 +/- 0.4 units of fluorescence/(100 microg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug.

Anti-proliferative and cytotoxic activities of the flavonoid isoliquiritigenin in the human neuroblastoma cell line SH-SY5Y.

Neuroblastoma is a common childhood cancer with high mortality. We evaluated the capacity of the flavonoid, isoliquiritigenin (4,2',4'-trihydroxychalcone; ISL) to inhibit cellular proliferation and migration in the human neuroblastoma cell line SH-SY5Y. Incubation of cultured SH-SY5Y cells with 20-100 µM ISL decreased cell confluency (15-70%) after 24 h incubation, while 10-100 µM ISL (24 h) depleted intracellular ATP stores (15-90% vs vehicle-treated control) after 24 h incubation. ISL-mediated cell toxicity did not involve intracellular caspase 3/7 activation, externalization of phosphatidylserine on the cell membrane or stimulation of TNF and IL-1ß release, all indicating that the flavonoid did not induce apoptosis. Pre-treatment of cells with necrostatin-1, a necroptosis inhibitor, significantly restored ATP levels (ATP levels increased 12-42%) in ISL-treated neuroblastoma cells indicative of enhanced viability. By contrast, RIP1 phosphorylation status remained unchanged in cells treated with ISL although the intracellular ratio of phosphorylated/total parental RIP1 increased after ISL treatment on SH-SY5Y cells indicating that ISL decreased levels of native RIP1. In addition, ISL treatment inhibited SH-SY5Y cell migration/proliferation in a scratch assay and arrested cell cycle transition by significantly decreasing the number of cells in G0/G1 phase and increasing populations by ~10% in S (primarily) and G2/M (lesser extent) phases. The intracellular ratio of phosphorylated/total ERK 1/2 and p38 remained unchanged after ISL treatment (up to 40 µM); ERK activation was only determined at ISL dose well above the experimental IC50 value as judged by ELISA analyses and this did not correlate with ISL cytotoxicity at lower dose <40 µM; Western blot assay confirmed the detection of phosphorylated (p-)ERK1/2 and (p-)p38 in ISL treated cells. Together the results suggest that ISL exerts anti-proliferative and cytotoxic activity on SHSY5Y cells through the loss of ATP, induction of cell cycle arrest, and cell death largely via a necroptotic mechanism in the absence of apoptotic activity.

Activity of isosteviol lactone on mitochondrial metabolism.

Isosteviol lactone (LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%), cytochrome c oxidase (10%), and NADH dehydrogenase (13%). Glutamate dehydrogenase was also a target for LAC, as it was 85% inhibited by 1 mM LAC. Cyclic voltammetry data showed that LAC, as well as ISO, does not undergo redox reactions under current experimental conditions. LAC (0.05-0.75 mM) inhibited the swelling dependent on the glutamate oxidation, 50% of the effect occurring at 0.5 mM LAC. Swelling supported by KNO(3) and valinomycin was also inhibited over all concentrations used of LAC and ISO, the effect being of a lower intensity for LAC, suggesting that the modification of the structure of ISO by lactonization diminished its interaction with the membrane. This could contribute to attenuation of the toxic effects described for ISO on mitochondrial function, such as those on respiratory chain enzymatic complexes and phosphorylating activity.

Effects of deltamethrin on functions of rat liver mitochondria and on native and synthetic model membranes.

Deltamethrin (DTM) is a synthetic pyrethroid insecticide used wideworld in agriculture, home pest control, protection of foodstuff, and disease vector control. It has widespread applications in Brazilian agriculture. The effects of DTM on mitochondrial respiratory parameters and on the organization of artificial and native membranes are described. DTM (200 nmol mg(-1) protein) on isolated liver mitochondria decreased oxygen consumption of both, state III and state IV, as well as the inner mitochondrial membrane potential (Deltapsi). Analysis of segments of the respiratory chain suggested that the DTM inhibition site is located between complex II and complex III. Mitochondrial swelling, energized or driven by the K+ diffusion potential using valinomycin, were partially inhibited by DTM (200 nmol mg(-1) protein). Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions, respectively, of dimyristoylphosphatidylcholine (DMPC) and native mitochondrial membranes, indicated that DTM shifts the midpoint phase transition to lower values, besides broadening the phase transition. DTM decreased the lipid order of DMPC bilayers, at temperatures lower than the transition temperature and also caused a disordering effect on native membranes. However at temperatures above the transition temperature, the pesticide increased the rigidity of the membrane. These results suggest that DTM causes perturbations in lipid-lipid and lipid-protein interactions, interferes in transport mechanisms operating at the membrane level, and causes alterations of membrane permeability and mitochondrial enzyme activities. These effects could be associated with the toxicity of deltamethrin.

Enolizable carbonyl and imino metabolites may act as endogenous sources of reactive oxygen species

Highly reactive oxyradicals and electronically excited triplet carbonyls can be generated in vitro by iron complexes and heme enzyme-catalyzed aerobic oxidation of synthetic or naturally occurring substances capable of enolization in aqueous medium. Monoenols and enamines, obtained by (alpha-methyne-carbonyl and -imine enolization, undergo dioxygen insertion and ultimately originate triplet species; e.g., isobutanal, 3-methylacetoacetone, Schiff bases. In turn, (alpha-hydroxy- and (alpha-aminocarbonyls (e.g., carbohydrates, 5-aminolevulinic acid) tautomerize to enediols and enolamines and yield oxyradicals, initiated by electron transfer to dioxygen, as polyphenols (e.g., 6-hydroxydopamine) and polyphenolamines do. Free radicals and excited species have been implicated in several normal and pathological processes. We here briefly review our contributions to this research area, emphasizing a possible in vivo prooxidant role for 5-aminolevulinic acid, the heme precursor accumulated in several porphyric disorders (e.g., lead poisoning, acut intermittent porphyria, tyrosinosis).