Study of the cytotoxic activity of Styrax camporum extract and its chemical markers,egonol and homoegonol

The benzofuran lignans egonol and homoegonol are found in all species of the genus Styrax. Since natural products are important sources of new anticancer drugs, this study evaluated the cytotoxic activity of a hydroalcoholic extract of the stems of S. camporum (SCHE) and their chemical markers, egonol (EG) and homoegonol (HE), against different tumor cell lines (B16F10, MCF-7, HeLa, HepG2, and MO59J). A normal human cell line (GM07492A) was included. Cytotoxic activity was evaluated at different treatment times (24, 48 and 72 h) using the XTT assay. More effective results were observed after 72 h of treatment. The lowest IC₅₀ values were found for the HepG2 cell line, ranging from 11.2 to 55.0 µg/mL. The combination of EG and HE exerted higher cytotoxic activity than SCHE or treatment with either lignan alone, with the lowest IC₅₀ (13.31 µg/mL) being observed for the MCF-7 line. Furthermore, treatment with these lignans was significantly more cytotoxic for some tumor cell lines compared to the normal cell line, GM07492A, indicating selectivity. These results suggest that these lignans may be used to treat cancer without affecting normal cells.     

Canine Gastrointestinal Pythiosis Treatment by Combined Antifungal and Immunotherapy and Review of Published Studies

Pythium insidiosum is an oomycete, a fungal like microorganism, which infects mammals, causing pythiosis in animals and humans, especially in tropical and subtropical regions around the world. The treatment for this infection is very difficult, and therapeutic options commonly comprise surgery, immunotherapy and antimicrobial drugs. The present report describes the clinical healing of a dog with gastrointestinal pythiosis by treatment with a combination of antifungals and immunotherapy, as well as reviews the cases reported in the literature that used some type of therapy for canine pythiosis. A 2.5-year-old male beagle initially showed sporadic vomiting episodes, and this symptom became more frequent 5 months after the onset of clinical signs. Celiotomy procedure found thickness of the stomach wall extending to the pylorus and duodenum. A biopsy was performed, and the diagnosis of pythiosis was made by mycological, histopathological analyses and molecular identification. Therapy was based on an association of terbinafine plus itraconazole during 12 months and immunotherapy for 2.5 months. The healing of the dog reported here allows us to propose the use of immunotherapy associated with antifungal therapy to treat canine gastrointestinal pythiosis. However, additional studies should be performed on a larger number of patients to establish a standard treatment protocol for canine pythiosis.     

Identification of <Emphasis Type="Italic">Pythium insidiosum</Emphasis> by Nested PCR in Cutaneous Lesions of Brazilian Horses and Rabbits

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.     

In Vivo Assessment of Genotoxic,Antigenotoxic and Anticarcinogenic Activities of Solanum lycocarpum Fruits Glycoalkaloidic Extract

The fruits of Solanum lycocarpum, known as wolf-fruit, are used in folk medicine, and because of that we have evaluated both the genotoxic potential of its glycoalkaloidic extract (SL) and its influence on the genotoxicity induced by methyl methanesulfonate. Furthermore, the potential blocking effect of SL intake in the initial stage of colon carcinogenesis in Wistar rats was investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as biomarker. The genotoxic potential was evaluated using the Swiss mice peripheral blood micronucleus test. The animals were treated with different doses of SL (15, 30 and 60 mg/kg b.w.) for 14 days, and the peripheral blood samples were collected at 48 h, 7 days and 14 days after starting the treatment. For antigenotoxicity assessment, MMS was administered on the 14^th day, and after 24 h the harvesting of bone marrow and liver cells was performed, for the micronucleus and comet assays, respectively. In the ACF assay, male Wistar rats were given four subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH, 40 mg/kg b.w.), twice a week, during two weeks to induce ACF. The treatment with SL (15, 30 and 60 mg/kg b.w.) was given for four weeks during and after carcinogen treatment to investigate the potential beneficial effects of SL on DMH-induced ACF. The results demonstrated that SL was not genotoxic in the mouse micronucleus test. In animals treated with SL and MMS, the frequencies of micronucleus and extensions of DNA damage were significantly reduced in comparison with the animals receiving only MMS. Regarding the ACF assay, SL significantly reduced the frequency of ACF induced by DMH.     

In vitro antiherpetic and antirotaviral activities of a sulfate prepared from Mimosa scabrella galactomannan

A chemically sulfated galactomannan (BRS) from seeds of Mimosa scabrella had in vitro antiviral activity against Herpes simplex virus 1 (HSV-1), but not against Simian rotavirus A/SA11 (SiRV-A/SA11). It was examined by ¹³C NMR spectroscopy, which showed the sulfate groups to be mainly at C-6 of galactose residues. BRS had a selective inhibition against HSV-1 during its attachment step, having an IC₅₀ lower than 2.5 μg/ml, determined by plaque reduction, and a selectivity index of greater than 181, suggesting that the antiviral effect is likely due to interactions between the virus and BRS, being influenced its overall surface charge.     

Fatty acids uptake and oxidation are increased in the liver of rats with adjuvant-induced arthritis

Severe rheumatoid cachexia is associated with pronounced loss of muscle and fat mass in patients with advanced rheumatoid arthritis. This condition is associated with dyslipidemia and predisposition to cardiovascular diseases. Circulating levels of triglycerides (TG) and free fatty acids (FFA) have not yet been consistently defined in severe arthritis. Similarly, the metabolism of these lipids in the arthritic liver has not yet been clarified. Aiming at filling these gaps this study presents a characterization of the circulating lipid profile and of the fatty acids uptake and metabolism in perfused livers of rats with adjuvant-induced arthritis. The levels of TG and total cholesterol were reduced in both serum (10–20%) and liver (20–35%) of arthritic rats. The levels of circulating FFA were 40% higher in arthritic rats, possibly in consequence of cytokine-induced adipose tissue lipolysis. Hepatic uptake and oxidation of palmitic and oleic acids was higher in arthritic livers. The phenomenon results possibly from a more oxidized state of the arthritic liver. Indeed, NADPH/NADP⁺ and NADH/NAD⁺ ratios were 30% lower in arthritic livers, which additionally presented higher activities of the citric acid cycle driven by both endogenous and exogenous FFA. The lower levels of circulating and hepatic TG possibly are caused by an increased oxidation associated to a reduced synthesis of fatty acids in arthritic livers. These results reveal that the lipid hepatic metabolism in arthritic rats presents a strong catabolic tendency, a condition that should contribute to the marked cachexia described for arthritic rats and possibly for the severe rheumatoid arthritis.     

Interaction Between Metals and Chelating Agents Affects Glutamate Binding on Brain Synaptic Membranes

The present study investigates the possible effects of Hg²⁺, Pb²⁺, and Cd²⁺ on [³H]-glutamate binding. To better understand the role of the thiol-disulfide status on the toxicity of such metals toward glutamatergic neurotransmission, we used three thiol chelating agents, 2,3-dimercaptopropanol (BAL), 2,3-dimercaptopropane 1-sulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (DMSA). Dithiotreitol (DTT) was tested for its ability to prevent metals-induced inhibition on [³H]-glutamate binding. Hg²⁺, Pb²⁺, and Cd²⁺ showed a concentration-dependent inhibition on [³H]-glutamate binding, and mercury was the most effective inhibitor. BAL did not prevent [³H]-glutamate binding inhibition by Hg²⁺, Cd²⁺, and Pb²⁺. However, DMPS and DMSA prevented the inhibition caused by Cd²⁺ and Pb²⁺, but not by Hg²⁺. DTT did not prevent the inhibition on [³H]-glutamate binding caused by 10 M Hg²⁺. In contrast, it was able to partially prevent [³H]-glutamate binding inhibition caused by 40 M Pb²⁺ and Cd²⁺. These results demonstrated that the heavy metals present an inhibitory effect on [³H]-glutamate binding. In addition, BAL was less effective to protect [³H]-glutamate binding inhibition caused by these metals than other chelating agents studied.     

Cadmium inhibits delta-aminolevulinate dehydratase from rat lung in vitro: interaction with chelating and antioxidant agents

The effect of cadmium (Cd(2+)) on delta-aminolevulinate dehydratase (delta-ALA-D) activity from rat lung in vitro was investigated. delta-ALA-D activity, a parameter for metal intoxication, has been reported as a target of Cd(2+) in different tissues. The protective effect of monotherapies with dithiol chelating (meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS)) or antioxidant agents (ascorbic acid, diphenyl diselenide (PhSe)(2), and N-acetylcysteine (NAC)) was evaluated. The effect of a combined therapy (dithiol chelatingxantioxidant agent) was also studied. Zinc chloride (ZnCl(2)) and dithiothreitol (DTT) were used to investigate the mechanisms involved in cadmium, chelating and antioxidant effects on delta-ALA-D activity. Cadmium inhibited rat lung delta-ALA-D activity at low concentrations. DTT (3mM), but not ZnCl(2) (100microM), protected the inhibition of enzyme activity caused by Cd(2+). Chelating agents were not effective in restoring the enzyme activity. DMPS and DMSA presented inhibitory effect on enzyme activity. DTT restored the inhibition caused by both chelating agents, but ZnCl(2) restored only the inhibitory effect induced by DMSA. These compounds caused a marked potentiation of delta-ALA-D inhibition induced by Cd(2+). ZnCl(2) did not restore inhibition of enzyme activity caused by Cd(2+) plus chelating agents. Conversely, DTT restored the inhibition induced by Cd(2+)/DMSA, but not by Cd(2+)/DMPS. Antioxidants were not effective in ameliorating delta-ALA-D inhibition induced by Cd(2+), whereas ascorbic acid potentiated the enzyme inhibition induced by this metal. A combined effect of Cd(2+)xDMPSx(PhSe)(2) and Cd(2+)xDMPSxNAC was observed. There was no combined effect of Cd(2+)xchelatorxantioxidants when DMSA was used. This study demonstrated that Cd(2+)inhibited delta-ALA-D activity and chelating and antioxidant agents, alone or combined, did not restore the enzyme activity. In contrast, these compounds potentiated the inhibition induced by Cd(2+) in rat lung.     

DMPS and N-acetylcysteine induced renal toxicity in mice exposed to mercury

Acute effects of mercuric chloride (HgCl₂) were evaluated on mice. Mice received a single dose of HgCl₂ (4.6 mg/kg, subcutaneously) for three consecutive days. Thirty minutes after the last injection with HgCl₂, mice received one single injection of 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetylcysteine (NAC) or diphenyl diselenide (PhSe)₂. DMPS, NAC and (PhSe)₂ were utilized as therapy against mercury exposure. At 24 h after the last HgCl₂ injection, blood, liver and kidney samples were collected. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K_-⁺ ATPase activities, thiobarbituric acid-reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid concentrations were evaluated. Plasma aspartate (AST) and alanine (ALT) aminotransferase activities, as well as urea and creatinine levels were determined. The group of mice exposed to Hg + (PhSe)₂ presented 100% of lethality. Exposure with HgCl₂ caused a decrease on the body weight gain and treatments did not modify this parameter. δ-ALA-D, AST and ALT activities, TBARS, ascorbic acid levels and NPSH (hepatic and erythrocytic) levels were not changed after HgCl₂ exposure. HgCl₂ caused an increase in renal NPSH content and therapies did not modify these levels. Mice treated with (PhSe)₂, Hg + NAC and Hg + DMPS presented a reduction in plasma NPSH levels. Creatinine and urea levels were increased in mice exposed to Hg + NAC, while Hg + DMPS group presented an increase only in urea level. Na⁺, K_-⁺ ATPase activity was inhibited in mice exposed to Hg + DMPS and Hg + NAC. In conclusion, therapies with (PhSe)₂, DMPS and NAC following mercury exposure must be better studied because the formation of more toxic complexes with mercury, which can mainly damage renal tissue.