In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis

In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC₉₀) = 0.125 μg/mL] and was superior to levofloxacin (MIC₉₀ = 1 μg/mL) and moxifloxacin (MIC₉₀ = 0.25 μg/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED₅₀) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED₅₀ values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration-time curve from 0-24 h (fAUC_0-24)/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7-11.4-fold and 1.4-fold greater than that of levofloxacin (6.44-8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis.     

Effects of a single exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on macro- and microstructures of feeding and drinking in two differently TCDD-sensitive rat strains

In rats, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes anorexia that may lead to fatal wasting but has hitherto been poorly characterized. Therefore, we studied in-depth feeding and drinking behaviors of TCDD-sensitive L-E rats for 5 (100 μg/kg; lethal dose) or 10 (10 μg/kg; sublethal) days and of TCDD-resistant H/W rats for 14 (100 or 1000 μg/kg; both sublethal) days postexposure to TCDD. The 1000-fold higher resistance of H/W rats to acute lethality of TCDD results from a mutation in their AH receptor (AHR). We split days into four (morning, daytime, evening, and night) or two (light/dark) circadian periods and took the repeated nature of the data into account. In L-E rats at 100 μg/kg, the feed intake dropped precipitously, due to reduced meal sizes. In H/W rats, the hypophagia remained moderate and stemmed from a reduced meal frequency. While the suppression in L-E rats peaked during the morning (at 100 μg/kg), the main effects in H/W rats were seen during the constant light or dark phases. Furthermore, chronologic data analysis revealed alterations in consecutive feeding and drinking patterns. Thus, striking differences were found between these strains in the timing and structure of consummatory behaviors, suggesting involvement of the AHR in these behaviors.     

Unexpected gender difference in sensitivity to the acute toxicity of dioxin in mice

The acute toxicity of the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) varies widely among species and strains. Previous studies in rats have established that females are approximately 2-fold more sensitive to TCDD lethality than males. However, there is a surprising gap in the literature regarding possible gender-related sensitivity differences in mice. In the present study, by using three substrains of TCDD-sensitive C57BL/6 mice and transgenic mice on this background, we demonstrated that: 1) in contrast to the situation in rats, female mice are the more resistant gender; 2) the magnitude of the divergence between male and female mice depends on the substrain, but can amount to over 10-fold; 3) AH receptor protein expression levels or mutations in the primary structure of this receptor are not involved in the resistance of female mice of a C57BL/6 substrain, despite their acute LD₅₀ for TCDD being over 5000 μg/kg; 4) transgenic mice that globally express the rat wildtype AH receptor follow the mouse type of gender difference; 5) in gonadectomized mice, ovarian estrogens appear to enhance TCDD resistance, whereas testicular androgens seem to augment TCDD susceptibility; and 6) the gender difference correlates best with the severity of liver damage, which is also reflected in hepatic histopathology and the expression of pro-inflammatory cytokines, especially IL-6. Hence, the two closely related rodent species most often employed in toxicological risk characterization studies, rat and mouse, represent opposite examples of the influence of gender on dioxin sensitivity, further complicating the risk assessment of halogenated aromatic hydrocarbons.     

Isoform distinct time-, dose-, and castration-dependent alterations in flavin-containing monooxygenase expression in mouse liver after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment

Flavin-containing monooxygenase (FMO) expression in male mouse liver is altered after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or castration. Because TCDD is slowly eliminated from the body, we examined hepatic Fmo mRNA alterations for up to 32 days following 10 or 64 μg/kg TCDD exposure by oral gavage in male C57BL/6J mice. Fmo2 mRNA was significantly induced at 1, 4, and 8 days whereas Fmo3 mRNA was also induced at 32 days relative to controls. Fmo3 mRNA levels exhibited a dose-dependent increase at 4, 8, and 32 days after exposure; Fmo1, Fmo4, and Fmo5 mRNA did not exhibit clear trends. Because castration alone also increased Fmo2, Fmo3, and Fmo4 mRNA we examined the combined effects of castration and TCDD treatment on FMO expression. A greater than additive effect was observed with Fmo2 and Fmo3 mRNA expression. Fmo2 mRNA exhibited a 3-5-fold increase after castration or 10 μg/kg TCDD exposure by oral gavage, whereas an approximately 20-fold increase was observed between the sham-castrated control and castrated TCDD-treated mice. Similarly, treatment with 10 μg/kg TCDD alone increased Fmo3 mRNA 130- and 180-fold in the sham-castrated and castrated mice compared to their controls respectively, whereas, Fmo3 mRNA increased approximately 1900-fold between the sham control and castrated TCDD-treated mice. An increase in hepatic Fmo3 protein in TCDD-treated mice was observed by immunoblotting and assaying methionine S-oxidase activity. Collectively, these results provide evidence for isoform distinct time-, dose-, and castration-dependent effects of TCDD on FMO expression and suggest cross-talk between TCDD and testosterone signal transduction pathways.     

Antinociceptive activity of Annona diversifolia Saff. leaf extracts and palmitone as a bioactive compound

Annonas are consumed as fresh fruits, but are also widely used in folk medicine for treating pain and other ailments. Antinociceptive properties of the Annona diversifolia ethanol crude extract were tested using the pain-induced functional impairment model in rat (PIFIR) and the writhing test in mice. The ethanol extract caused a 25% recovery of limb function in rats; this response was significant and dose-dependent. Furthermore, this extract produced a similar antinociceptive response (ED₅₀ = 15.35 mg/kg) to that of the reference drug tramadol (ED₅₀ = 12.42 mg/kg) when evaluated in the writhing test in mice. Bio-guided fractionation yielded hexane and acetone active fractions from which the presence of palmitone and flavonoids was respectively detected. Palmitone produced an antinociceptive response with an ED₅₀ = 19.57 mg/kg in the writhing test. Antinociceptive responses from ethanol extract and tramadol were inhibited in the presence of either naloxone (1 mg/kg, s.c.)—an antagonist of endogenous opioids—or WAY100635 (0.8 mg/kg, s.c.)—a 5-HT_1A serotonin receptor antagonist. These results provide evidence that A. diversifolia possesses antinociceptive activity, giving support to their traditional use for treatment of spasmodic and arthritic pain. In addition, our results suggest the participation of endogenous opioids and 5-HT_1A receptors in this antinociceptive response.     

Toxicity of Jatropha curcas phorbol esters in mice

Phorbol esters are the main toxins in Jatropha curcas seed and oil. The aim of this study was to assess the acute toxicity of phorbol esters given by intragastric administration and to determine the LD₅₀ for Swiss Hauschka mice. The LD₅₀ and 95% confidence limits for male mice were 27.34 mg/kg body mass and 24.90–29.89 mg/kg body mass; and the LD₅ and LD₉₅ were 18.87 and 39.62 mg/kg body mass, respectively. The regression equations between the probits of mortalities (Y) and the log of doses (D) was Y = −9.67 + 10.21 log (D). Histopathological studies on the organs from the dead mice showed: (1) no significant abnormal changes in the organs at the lowest dose (21.26 mg/kg body mass) studied, (2) prominent lesions mainly found in lung and kidney, with diffused haemorrhages in lung, and glomerular sclerosis and atrophy in kidney at doses ?32.40 mg/kg body mass, and (3) multiple abruption of cardiac muscle fibres and anachromasis of cortical neurons at the highest dose of 36.00 mg/kg body mass. The results obtained would aid in developing safety measures for the Jatropha based biofuel industry and in exploiting the pharmaceutical and agricultural applications of phorbol esters.     

Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice

 The dose-response relationship of cytochrome P4501b1 (Cyp1b1) and Cyp1a1 induction in livers of TCDD-treated female C57BL/6J and DBA/2J mice are described. The animals were treated i.p. with 0.001, 0.01, 0.1, 1, 10 and 50 μg TCDD/kg for 24 h, and Cyp1b1 and Cyp1a1 mRNA expression was analyzed by RT-PCR. In the livers of both mouse strains, the Cyp1b1 and Cyp1a1 mRNA content was increased after TCDD exposure in a dose-dependent manner. These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice, although Cyp1a1 was more responsive to TCDD than Cyp1b1 in both strains. The calculated ED₅₀ values for Cyp1b1 and Cyp1a1 induction in livers of TCDD-treated C57BL/6J mice were 1.3 and 0.08 μg TCDD/kg, respectively. The corresponding values for half-maximal induction response in livers of DBA/2J mice were 3.4 μg TCDD/kg for Cyp1b1 and 1.5 μg TCDD/kg for Cyp1a1. These results show that Cyp1b1 mRNA expression is less inducible by TCDD than Cyp1a1. Both genes are highly inducible in TCDD-responsive C57BL/6J mice expressing the high affinity arylhydrocarbon receptor (Ah receptor), suggesting that Cyp1b1, like Cyp1a1, is a potential Ah receptor-regulated gene. Received: 8 December 1995/Accepted: 6 February 1996     

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50–300 μg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 μg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H₂O₂ toxicity was observed only when cells were treated with EO during and after exposure to H₂O₂. With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 μg/mL EO. In conclusion, EO at concentrations up to 300 μg/mL or doses up to 400 mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H₂O₂.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

Purification, characterization and cytotoxicity of malanin, a novel plant toxin from the seeds of Malania oleifera

Malanin, a novel plant toxin with a molecular weight of 61,875 Da and an isoelectric point of 5.5, was isolated from Malania oleifera seeds by homogenization, ammonium sulfate precipitation and hydrophobic interaction chromatography (HIC). It is a glycoprotein with two chains, chain-A and chain-B, which are crosslinked by one or more disulfide bonds. The N-terminal amino-acid sequences of malanin are DETXTDEEFN (X was commonly C) in chain-B, and DYPKLTFTTS in chain-A. Malanin exhibited highly cytotoxic activities against cancer cell lines (HeLa, PC-12, MCF-7, K562) and non-cancer cell lines (Vero and MDCK), producing IC₅₀ values of 0.15 ± 0.08, 7.71 ± 0.24, 11.20 ± 0.02, 15.80 ± 0.09, 2.79 ± 0.05 and 3.92 ± 0.01 nM, respectively. It significantly inhibited the growth of HeLa cells through cell-cycle arrest at S phase and induced an apoptotic response. LD₅₀ values were determined in ICR mice, which were found to be 26.22 μg/kg and 43.11 mg/kg by i.p. and i.g. respectively. Thus, malanin is amongst the most potent toxin of plant origin.     

Survivors of soman poisoning: recovery of the soman LD50 to control value in the presence of extensive acetylcholinesterase inhibition

Initially, mice were pretreated with atropine (17.4 mg/kg; IP) and the oxime reactivator HI-6 (50 mg/kg; IP) 5 min prior to an injection of soman (287 g/kg, SC); approximately 2.1 × LD₅₀ dose). More than 95% of the mice survived this dose of soman with atropine and HI-6 pretreatment. In these survivors of soman poisoning the return of the soman LD₅₀ value to control value (124 g/kg, SC) was determined at various times after the initial soman exposure. Mice which survived exposure to a lethal dose of soman by pretreatment with atropine and HI-6 were sensitized to the lethal effects of soman upon re-exposure. The SC soman LD₅₀ at 4 h, after surviving the initial soman exposure, was 20 g/kg. The normal soman LD₅₀ (as evidenced by a LD₅₀ value which was not significantly different from the control value) returned within 4 days, at which time there was still extensive acetylcholinesterase inhibition in all brain regions (striatum, pons-medulla, cerebellum, hypothalamus, hippocampus), diaphragm and erythrocytes. Serum carboxylesterase recovered to control levels within 48 h, whereas liver carboxylesterase activity was not inhibited following the initial soman exposure. The results demonstrate that there is an excess of acetylcholinesterase which is required for normal response in the toxicological sense.     

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC₅₀ value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c_max at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c_max. The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K_i values were determined. All K_i values exceeded c_max by at least a factor of 10-fold. According to FDA regulations 1 > c_max/K_i > 0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.     

Post-exposure therapy of inhalational anthrax in the common marmoset

The aim of this study was to compare the pharmacokinetics and efficacy of ciprofloxacin as post-exposure therapy against inhalational anthrax in the common marmoset (Callithrix jacchus) with other non-human primate models in order to determine whether the marmoset is a suitable model to test post-exposure therapies for anthrax. Pharmacokinetic (PK) and efficacy studies with ciprofloxacin were performed in the marmoset. Ciprofloxacin plasma pharmacokinetics were determined in six animals in separate single-dose and multiple-dose studies and were analysed by high-performance liquid chromatography (HPLC). A separate group of marmosets was exposed to ca. 100× the 50% lethal dose (LD₅₀) of Bacillus anthracis Ames strain by the airborne route. On Day 5 of a twice-daily dosing regimen of 17.5 mg/kg, the ciprofloxacin half-life (t_1/2), maximum drug concentration (C_max) and area under the concentration-time curve (AUC) in marmoset plasma were 1.9 h, 2.1 μg/mL and 7.9 μg/mL/h, respectively. Naïve untreated control animals succumbed to infection by Day 9. All animals treated with ciprofloxacin, started on the day of exposure and continued for 10 days, remained healthy during the treatment period. Two antibiotic-treated animals (33%) died after withdrawal of antibiotic therapy, attributed to the germination of residual spores. In conclusion, in many respects the marmoset appears to respond to B. anthracis in a similar way to the macaque, suggesting that this small non-human primate is an acceptable, practical alternative model for the evaluation of medical countermeasures against respiratory anthrax infection.     

Isolation and pharmacological effects of leptoxin, a novel proteic toxin from Leptodactylus pentadactylus skin secretion

The in vivo and in vitro pharmacological effects of leptoxin, one of the most lethal protein toxins known at present date (LD₅₀ 0.5 ± 0.03 μg/kg i.v., mice) isolated from Leptodactylus pentadactylus skin secretion, were studied. In rats, leptoxin (1.0 μg/kg, i.v.) induced cardiorespiratory collapse with abundant tracheal secretion followed by sudden death. The cardiovascular shock, pulmonary edema and mortality were not prevented by pretreating the animals with effective doses of pharmacological blockers, i.e., atropine with or without bilateral vagotomy, phentolamine, propranolol, hexamethonium, captopril, dexamethasone, indomethacin, L-NAME, promethazine, Ginkgolide BN-52021 or tezosentan. Pulmonary macroscopic examination revealed increased tracheobronchial secretion, hemorrhagic areas and edema. Microscopic examination showed intense vascular congestion, alveolar and septal interstitial hemorrhage and alveolar edema, without infiltrated inflammatory cells. Leptoxin increased pulmonary index (0.67 ± 0.09 vs. 1.55 ± 0.24; p < 0.05) and the Evans blue concentration in the bronchoalveolar fluid (1.24 ± 0.17 vs. 4.17 ± 1.47 μg/μL; p < 0.01) and in the lung parenchyma (40.73 ± 3.27 vs. 65.33 ± 4.51 μg/μL; p < 0.03). Leptoxin increased the pulmonary perfusion pressure from 13.7 ± 5.3 to 54.0 ± 6.3 mmHg. It also induced a vasoconstrictor effect in the perfused mesenteric vascular bed that could be explained by a hyperreactivity to phenylephrine. Thus, the results suggest that leptoxin-induced death occurs by acute pulmonary edema due to increased microvascular pulmonary pressure evoked by direct vasoconstriction. Despite its strong toxicity, the role of leptoxin in L. pentadactylus skin remains unknown.