Primary and secondary clarithromycin, metronidazole, amoxicillin and levofloxacin resistance to Helicobacter pylori in southern Poland

The aim of this study was to assess the primary and secondary resistance of H. pylori strains cultured from adult patients of the Ma?opolska region of Poland, mainly of Kraków and the surrounding areas, to antibacterial agents (amoxicillin, clarithromycin, metronidazole and levofloxacin). In total, 115 H. pylori strains were isolated, of which 90 strains originated from patients who had never been treated for H. pylori infection, while the remaining 25 were isolated from patients in whom eradication of the infection failed after treatment. All tested H. pylori strains were susceptible to amoxicillin. Forty-four percent of strains isolated were resistant to metronidazole. The primary and secondary resistance to this antimicrobial chemotherapeutic reached 37% and 72% (p = 0.002), respectively. In total, 34% of strains were resistant to clarithromycin, and the ratio of strains with secondary resistance was significantly greater than that of the strains with primary resistance (80% vs. 21%, p < 0.001). The double resistance to both metronidazole and clarithromycin was confirmed in 23% of H. pylori strains. Five percent of H. pylori strains were resistant to levofloxacin, while primary and secondary resistance to this drug accounted for 2% and 16% (p = 0.006), respectively. In total, 4% of H. pylori strains were simultaneously resistant to metronidazole, clarithromycin and levofloxacin. Thus, the high resistance to metronidazole and clarithromycin excludes the possibility of using these drugs for treatment of H. pylori infection without earlier antibiogramming. Levofloxacin, as a drug of high efficacy against H. pylori, should be reserved for an “emergency” therapy and used in a limited capacity in order to preserve its potent antimicrobial activity. The Polish Society of Gastroenterology recommends levofloxacin as a third-line therapy [14].     

Inhibitory Effects of 4-Guanidinobutyric Acid against Gastric Lesions

This study examined the inhibitory effects of 4-guanidinobutyric acid (4GBA), an alkaloid, against gastric lesions by assessing the inhibition of Helicobacter pylori (H. pylori) and gastric cancer cells. Acute and chronic gastritis were also observed using HCl/ethanol (EtOH) and indomethacin-induced gastric lesion models, respectively. 4GBA inhibited the growth of H. pylori in a dose dependent manner, and showed acid-neutralizing capacity. In the pylorus ligated rats, 4GBA decreased the volume of gastric secretion and gastric acid output slightly, and increased the pH. 4GBA at a dose of 100 mg/kg reduced the size of HCl/EtOH-induced gastric lesions (70.8%) and indomethacin-induced gastric lesions (38.8%). The antigastritic action of 4GBA might be associated with the acid-neutralizing capacity, anti-H. pylori action, and decreased volume of gastric secretion. These results suggest that 4GBA might be useful in the treatment and/or protection of gastritis.     

Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries

IntroductionThe vasomotor response of the coronary artery is altered by hypoxia–reoxygenation (H–R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H–R injury.MethodsPorcine coronary arterial rings were isolated and placed in Krebs–Henseleit (K–H) solution. Rings were exposed to normoxic conditions (control group) and two different H–R conditions: the first induced by a 95% N₂–5% CO₂ gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 °C) hypoxia–reoxygenation in an air-tight beaker filled with K–H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K–H solution aerated with a 95% O₂–5% CO₂ mixture under normothermic (37 °C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferroni's post-test.ResultsDistal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p < 0.001) from the values for normoxic rings (81.0 ± 1.0%, n = 30) after 40-min H–R (50.5 ± 5.3%, n = 30), 60-min H–R (32.1 ± 3.5%, n = 30), 24-hours hypothermic H–R (56.0 ± 2.3%, n = 30) and after 48-hours hypothermic H–R (38.5 ± 5.1%, n = 30).ConclusionsThe model employing 40-min normothermic H–R is as effective as 24-hours hypothermic H–R, and 60-min normothermic H–R as 48-hours hypothermic H–R for studying the deleterious effects of H–R on EDHF-mediated relaxation.     

The role of small molecular weight compounds to increase vacuolation induced by VacA toxin in vitro

VacA is a vacuolation protein toxin secreted by Helicobacter pylori. Many compounds have been implicated in the regulation of VacA toxin activity. In this study, regulation of cell vacuolation induced by VacA was observed with the addition of glycine, glycine hydrochloride, xylitol, and taurine by neutral red dye uptake assay using gastric human epithelial cell cultures. Glycine, xylitol, and taurine increased cell vacuolation significantly after 48 h (p < 0.05), with their effect apparent in a wide concentration range (0.2 mM to about 100 mM). Changes were sharp in respect of concentration and showed little dose–response characteristics. In contrast, upregulation of glycine hydrochloride on cell vacuolation in weak acidic extracellular pH was much retarded with VacA activity not initiated until 72 h. In addition, our results showed that cell vacuolation was highest when the pH was 6.8. The increase in vacuolation was gentle in weak acidic extracellular pH and the increase dose-dependent with a Pearson correlation coefficient (r) of 0.986 from 0.2 to 6.25 mM. In this concentration range and at the same time point, the pH decrease was negatively correlated with vacuolating activity (r = 0.922, p < 0.01). In conclusion, our study showed that three small molecular compounds can increase vacuolation induced by VacA toxin in vitro.     

Healthy children's identification and risk perception of medicines in England

BackgroundChildren’s understanding of medicines has an impact on their behavior toward those medicines, and yet there has been a paucity of studies exploring this area.ObjectivesTo assess children’s ability to identify and to explore their risk perceptions of medicines.MethodsOne hundred eighty-two children aged 4 to 11 years at 2 primary schools in England completed a worksheet containing photos of foods and pharmaceutical products. Children were asked to identify what the picture showed and classify it as “good for them,” “bad for them,” or “sometimes good/sometimes bad for them.” Responses were marked as correct if they identified an item without the need for exact identification. Where an item was correctly identified, risk perception was analyzed.ResultsChildren correctly identified 5 of the 7 pictures as a form of medicine (mean = 5.10, standard deviation = 1.51), and identification was positively correlated with age (ρ = 0.59, P < .001). A greater percentage of children correctly identified bicolored capsules (86.3% correct, 95% confidence interval [CI] = 81.3-91.3) as medicines than either white (71.4% correct, 95% CI = 64.9-78) or pink tablets (33.5% correct, 95% CI = 26.7-40.4). There was a significant shift with age in the perceptions of the children as they changed from reporting that medicines were good for them to reporting that they were sometimes good and sometimes bad for them. This held for all medicines (χ² tests, P < .05) except for the cream and the inhaler.ConclusionsAs children get older, they become better at identifying medicines, and they become more likely to see their potential risks.     

Characterization of fasted human gastric fluid for relevant rheological parameters and gastric lipase activities

PurposeTo characterize human gastric fluid with regard to rheological properties and gastric lipase activity. In addition, traditional physicochemical properties were determined.MethodsFasted HGA were collected from 19 healthy volunteers during a gastroscopic examination. Rheological characterization of the aspirates was conducted on a TA AR-G2 rheometer, using cone and plate geometry. Lipase activity was measured by continuous titration of released free fatty acid from tributyrate. Further, pH, osmolality, buffer capacity, and surface tension were measured and the total protein content and bile salt level were determined using assay kits.ResultsRheological examination of HGA showed non-Newtonian shear-thinning behavior with predominant elastic behavior in the linear range. The apparent viscosity was measured to be in the range of 1.7–9.3 mPa s at a shear rate of 50 s⁻¹. The FaSSGF and HCl pH 1.2 have no shear-thinning properties and showed lower viscosity (1.1 mPa s at 50 s⁻¹). The observed viscosity of the HGA will decrease the intrinsic dissolution rate of drugs. The activity of the gastric lipase was 7.4 ± 4.0 U/mL (N = 6, n = 3) and 99.0 ± 45.3 U/mL (N = 19, n = 3) at pH 2.8 and 5.4, respectively. pH, surface tension, buffer capacity, bile salt concentration, and osmolality were measured and compared with literature data.ConclusionThe rheological behavior and the mean apparent viscosity of HGA are significantly different from that of water and should therefore be considered important during development of gastric simulated media. Further, the activity of the HGL is active even under fasted gastric conditions and might contribute to the digestion and emulsification of lipid-based drug delivery systems in the entire gastrointestinal tract. HGL should therefore be considered in gastric evaluation of lipid-based drug delivery systems.     

Worldwide dissemination of acquired carbapenem-hydrolysing class D β-lactamases in Acinetobacter spp. other than Acinetobacter baumannii

The aim of this study was to identify acquired OXA-type carbapenemases in Acinetobacter spp. other than Acinetobacter baumannii. From a total of 453 carbapenem-susceptible and -resistant Acinetobacter isolates collected worldwide, 23 were positive for bla_OXA genes by multiplex PCR. These isolates were identified as Acinetobacter pittii (n = 18), Acinetobacter nosocomialis (n = 2), Acinetobacter junii (n = 1) and Acinetobacter genomic species 14TU/13BJ (n = 2). The bla_OXA genes and associated insertion sequence (IS) elements were sequenced by primer walking. In 11 of these isolates, sequencing of the PCR products revealed that they were false-positive for bla_OXA. The remaining 12 isolates, originating from Europe, Asia, South America, North America and South Africa, harboured OXA-23 (n = 4), OXA-58 (n = 5), OXA-40-like (n = 1) and OXA-143-like (n = 1); one A. pittii isolate harboured both OXA-23 and OXA-58. IS elements were associated with bla_OXA in 10 isolates. OXA multiplex PCR showed a high degree of false-positive results (47.8%), indicating that detection of bla_OXA in non-baumannii Acinetobacter spp. should be confirmed using additional methods.     

Perfil de uso de los diferentes tipos de nutrición artificial en un hospital de agudos y de crónicos

ObjectiveTo evaluate the use of parenteral, enteral, and mixed nutrition in one acute and one chronic hospital.DesignRetrospective, non-randomised, observational study.Study sitesSouth Seville Health Area: Acute Hospital (H1) and Chronic Hospital (H2) with 447 and 84 beds, respectively.We analysed all episodes of artificial nutrition administered in a 6-month period. Exclusion criteria included: age <18 years, oral supplements, and peripheral nutrition.ResultsArtificial nutrition was used in a total of 568 episodes: 406 were enteral nutrition, 162 were parenteral nutrition, constituting 4.95%, 3.54% and 1,41% of all hospitalisations, respectively. Enteral nutrition was more common at H2 hospital (n = 219, 15.5/100 hospitalisations) and parenteral nutrition was more commonly used at H1 (n = 155, 6.96/100 hospitalisations), with the ICU providing the majority of treatments (43.8%).Mixed nutritional support was used in 68 patients (0.59% of all cases), and was most commonly used in the surgery department (n = 32, P<.001). The most commonly used enteral formula was the special diabetes diet; 41.2% at H1 and 46.6% at H2. Patient mortality with enteral nutrition was 37% at H1, 63% at H2, and was correlated with age (OR = 1.025, 95% CI: 1.006-1.046, P<.05), male sex (OR = 1.612, 95% CI: 1.023-2.540, P<.05), and time in ICU (OR = 49.379, 95% CI: 11.971-203.675, P<.01).ConclusionsEnteral nutrition was more frequently used in both the acute and chronic hospitals. Parenteral nutrition and mixed nutritional support were used almost exclusively at the acute hospital.     

Formulation,optimization and evaluation of transferosomal gel for transdermal insulin delivery

The present study deals with the development of transferosomal gel containing insulin by reverse phase evaporation method for painless insulin delivery for use in the treatment of insulin dependent diabetes mellitus. The effect of independent process variables like ratio of lipids (soya lecithin:cholesterol), ratio of lipids and surfactants, and ratio of surfactants (Tween 80:sodium deoxycholate) on the in vitro permeation flux (μg/cm²/h) of formulated transferosomal gels containing insulin through porcine ear skin was optimized using 2³ factorial design. The optimal permeation flux was achieved as 13.50 ± 0.22 μg/cm²/h with drug entrapment efficiency of 56.55 ± 0.37% and average vesicle diameter range, 625–815 nm. The in vitro insulin permeation through porcine ear skin from these transferosomal gel followed zero-order kinetics (R² = 0.9232–0.9989) over a period of 24 h with case-II transport mechanism. The in vitro skin permeation of insulin from optimized transferosomal gel by iontophoretic influence (with 0.5 mA/cm² current supply) also provided further enhancement of permeation flux to 17.60 ± 0.03 μg/cm²/h. The in vivo study of optimized transferosomal gel in alloxan-induced diabetic rat has demonstrated prolonged hypoglycemic effect in diabetic rats over 24 h after transdermal administration.     

Percutaneous absorption and metabolism of [14C]-ethoxycoumarin in a pig ear skin model

The biotransformation of chemicals by the skin can be a critical determinant of systemic exposure in humans following dermal absorption. Pig ear skin, which closely resembles human skin, is a candidate ex vivo alternative model for the investigation of xenobiotics penetration and metabolism. We developed an ex vivo pig ear skin model and explored its absorption, diffusion and metabolic capabilities using the model compound ¹⁴C-ethoxycoumarin (7-EC). Experimentations were undertaken on pig ear skin explants after application of various ¹⁴C-EC doses. Diffusion was quantified as well as the production of 7-EC metabolites resulting from phases I and II enzyme activities, using radio-HPLC. After 48 h, most of the radioactivity was absorbed and was recovered in culture media (70%) or in the skin itself (10%). 7-EC metabolites were identified as 7-hydroxycoumarin (OH–C) and the corresponding sulfate (S–O–C) and glucuronide (G–O–C) conjugates. Their formation followed Michaelis–Menten kinetics with saturation reached around 100 μM of 7-EC.Results demonstrate that dermal absorption as well as phases I and II enzymatic activities of pig skin are both functional. This model should represent a valuable alternative for the study of the transdermal exposure to chemicals, combining a functional dermal barrier and active biotransformation capabilities.     

CYP2B6 haplotype and biological factors responsible for hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral therapy

Data on the pharmacogenetic markers of CYP2B6 and biological factors associated with hepatotoxicity in HIV-infected patients receiving an efavirenz-based antiretroviral therapy (ART) regimen are very limited. A total of 134 HIV-infected Thai adults were prospectively enrolled to receive a once-daily regimen of efavirenz 600 mg/tenofovir/lamivudine. Seven single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped using real-time PCR. At 12 weeks after ART, plasma efavirenz concentrations at 12 h after dosing were measured. The mean ± standard deviation patient age was 37 ± 8 years, and 77.6% were male. The median (IQR) CD4 count was 43 cells/mm³ (17–105 cells/mm³). Eighteen patients (13.4%) had positive anti-HCV and 5 patients (3.7%) had positive HBsAg. The frequencies of heterozygous/homozygous mutants of each SNP were 64C>T (11%), 499C>G (0%), 516G>T (55%), 785A>G (63%), 1375A>G (0%), 1459C>T (3%) and 21563C>T (62%). The three most frequent haplotypes identified included *1/*6 (40.3%), *1/*1 (34.3%) and *6/*6 (8.2%). The median (IQR) plasma efavirenz concentration was 2.3 mg/L (1.4–3.7 mg/L). At 24 weeks, median (IQR) serum ALP was 98 mg/dL (73–133 mg/dL) and direct bilirubin was 0.11 mg/dL (0.10–0.19 mg/dL). The proportion of grade 1 and grade 2 elevated serum ALP was 12.7% and 1.5%, respectively. By multivariate analysis, factors associated with high ALP, total bilirubin and direct bilirubin included CYP2B6 haplotype *6/*6, high serum ALP at Week 0 and positive anti-HCV (all P < 0.05). In summary, HIV-infected patients with the pharmacogenetic marker ‘CYP2B6 haplotype *6/*6’ may have increased susceptibility to hepatotoxicity with efavirenz-based ART.     

Gardenia jasminoides Ellis ethanol extract and its constituents reduce the risks of gastritis and reverse gastric lesions in rats

In this study we investigated the effects of Gardenia jasminoides Ellis (GJE) extract and its constituents, such as ursolic acid and genipin, on gastritis in rats and the growth of human gastric cancer cells. The GJE extract, ursolic acid and genipin showed the acid-neutralizing capacities, the antioxidant activities, and the inhibitory effects on the growth of Helicobacter pylori (H. pylori), which are almost equivalent to positive control compounds. In addition, the GJE extract and ursolic acid had cytotoxic activity against AGS and SUN638 gastric cancer cells. The genipin and ursolic acid inhibited significant HCl/ethanol-induced gastric lesions. Taken together, GJE extract and its constituents might have antigastritic activities, associated with the antioxidant activities, acid-neutralizing capacities, and anti-H. pylori action. Also, we could suggest that genipin and ursolic acid may be useful for the treatment and/or protection of gastritis.     

Corticosteroids therapy and peptic ulcer disease in nephrotic syndrome patients

AIMS

Whether corticosteroids induce peptic ulcer disease (PUD) remains uncertain. The study evaluated and compared the occurrence of PUD between nephrotic patients receiving oral prednisolone therapy and nephritic patients without steroid therapy.

METHODS

The prospective case control study compared 60 nephrotic syndrome patients who received 60 mg daily prednisolone therapy for 3 months with 30 age-and sex-matched nephritic patients without steroid therapy. Each patient underwent endoscopic examination and tissue and blood sampling before and after the study. Examined parameters included Helicobacter pylori (H. pylori) infection, and gastric and serum prostaglandin (PG) E₂ and thromboxane (TX) B₂ concentrations. The primary endpoint was the occurrence of endoscopic peptic ulcers between the two groups, while the secondary end point was the occurrence of ulcer complications.

RESULTS

The two groups were comparable in sex, age, smoking habits, alcohol drinking, past history of PUD, H. pylori infection rate and serum creatinine. There were no differences in the occurrence of endoscopic peptic ulcers (1.6% vs. 3.3%) and ulcer complications (0% vs. 0%), pre-therapy gastric PGE₂, and pre- and post-therapy gastric TXB₂, serum PGE₂ and serum TXB₂ between the two groups. However, there was significantly lower post-therapy gastric PGE₂ concentrations in the prednisolone group.

CONCLUSIONS

Three-month therapy with 60 mg daily prednisolone caused few endoscopic ulcers (1.6%) and no ulcer complications in nephrotic patients. Corticosteroids therapy did not increase PUD in nephrotic syndrome patients [odds ratio 0.492 with 95% confidence interval (CI) 0.03, 8.142, P= 0.620]. Further larger studies are needed to clarify the role of corticosteroids in PUD.     

Analysis of Nifedipine Absorption from Soft Gelatin Capsules Using PBPK Modeling and Biorelevant Dissolution Testing

Delayed absorption of nifedipine when administered as a 20 mg immediate release soft gelatin capsule to fasted volunteers has been reported. Physiologically based pharmacokinetic (PBPK) modeling and in vitro dissolution data were used to explore our hypothesis that at high doses of nifedipine it precipitates in the stomach. Plasma concentration-time profiles following different doses of nifedipine were simulated using commercial PBPK software and compared to in vivo data. In vitro dissolution tests were performed with Adalat® 10 mg capsules in different volumes of fasted state simulated gastric fluid (FaSSGF). The discrepancy in plasma concentration-time profiles between the different nifedipine doses could be well simulated, assuming protracted dissolution for the 20 mg dose. Nifedipine release from one Adalat® 10 capsule in 250 or 500 mL FaSSGF was completed within 15 min whereas when release from two capsules, corresponding to 20 mg nifedipine, was studied in 250 mL FaSSGF, a maximum of about 75% drug dissolved was observed after 15 min followed by a decline in the % dissolved to a final value of approximately 40%. Based on the in silico and in vitro results it can be concluded that the observed prolongation in nifedipine absorption following the 20 mg dose was likely caused by nifedipine precipitation in human stomach.     

Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice

The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3 ± 1.5 × 10⁶ AECs. Of these 61 ± 13% and 43 ± 5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4 ± 1.1 × 10⁶ AECs. Flow cytometry of lectin bound AECs determined 91 ± 7% and 87 ± 5% as positive for Helix pomatia and Maclura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251 ± 80 pmol/4 h × 1.5 × 10⁶ and the production of androstenedione proceed at 243.5 ± 344.4 pmol/24 h × 1.5 × 10⁶ AECs.However, 6-α, 6-β and 16-β-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence.     

Ethanolic extract of roots from Arctium lappa L. accelerates the healing of acetic acid-induced gastric ulcer in rats: Involvement of the antioxidant system

We evaluate the curative efficacy of the ethanolic extract (EET) of roots from Arctium lappa (bardana) in healing of chronic gastric ulcers induced by 80% acetic acid in rats and additionally studies the possible mechanisms underlying this action.Oral administration of EET (1, 3, 10 and 30 mg/kg) reduced the gastric lesion area in 29.2%, 41.4%, 59.3% and 38.5%, respectively, and at 10 mg/kg promoted significant regeneration of the gastric mucosa, which was confirmed by proliferating cell nuclear antigen immunohistochemistry. EET (10 mg/kg) treatment did not increase the gastric mucus content but restored the superoxide dismutase activity, prevented the reduction of glutathione levels, reduced lipid hydroperoxides levels, inhibited the myeloperoxidase activity and reduced the microvascular permeability. In addition, EET reduced the free radical generation and increased scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals in vitro. Furthermore, intraduodenal EET (10 and 30 mg/kg) decreased volume and acidity of gastric secretion. Total phenolic compounds were high in EET (Folin–Ciocalteau assay) and the analysis by liquid chromatography-mass spectrometry revealed that the main compounds present in EET were a serie of hydroxycinnamoylquinic acid isomers. In conclusion, these data reveal that EET promotes regeneration of damaged gastric mucosa, probably through its antisecretory and antioxidative mechanisms.     

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM_2.5) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM_2.5 in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM_2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM_2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24 h of exposure (p < 0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48 h of exposure (p < 0.01), while those genes expressions were significantly reduced after 72 h of exposure, at which time the expression of p21 was predominant (p < 0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM_2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM_2.5-induced damage to the trachea and lung tissue was time-dependent.     

Follow-up to the pre-validation of a harmonised protocol for assessment of CYP induction responses in freshly isolated and cryopreserved human hepatocytes with respect to culture format,treatment, positive reference inducers and incubation conditions

We have compared induction responses of human hepatocytes to known inducers of CYP1A2, CYP2B6, CYP2C and CYP3A4/5 to determine whether the culture format, treatment regimen and/or substrate incubation conditions affected the outcome. CYP induction responses to prototypical inducers were equivalent regardless of pre-culture time (24 h or 48 h), plate format (60 mm or 24-well plates) used or whether CYP activities were measured in microsomes or whole cell monolayers. Fold-induction of CYP3A4/5 by 1000 μM PB and 10 μM RIF were equivalent. In contrast, the fold-induction of CYP2B6 by PB was 3-fold higher that by 10 μM RIF. In addition to inducing CYP1A2, 50 μM OME also induced CYP3A4/5 in 50% of the donors tested. CYP2B6 was induced in 14 out of 21 donors by BNF; however CYP3A4/5 was unaffected by BNF in these donors. In order to confirm that donor-to-donor variation was not due to inter-laboratory differences, the induction responses of 5 different batches of cryopreserved human hepatocytes were compared in two different laboratories. The induction of CYP1A2, CYP2B6 and CYP3A4 measured in our laboratory were equivalent to those obtained by the commercial companies, proving good between-laboratory reproducibility.In conclusion, there is some flexibility in the treatment and incubation protocols for classical CYP induction assays on human hepatocytes. Both RIF and PB are suitable positive control inducers of CYP3A4/5 but PB may be more appropriate for CYP2B6 induction. BNF may be more appropriate for CYP1A2 induction than OME since, in contrast to the latter, it does not induce CYP3A4. Induction responses using hepatocytes from the same donor but in different labs can be expected to be similar. The good reproducibility of induction responses between laboratories using cryopreserved hepatocytes underlines the usefulness of these cells for these types of studies.     

A validated bioanalytical method in mouse,rat, rabbit and human plasma for the quantification of one of the steroid glycosides found in Hoodia gordonii extract

Hoodia gordonii extract contains steroid glycosides, fatty acids, plant sterols and polar organic material. Certain steroid glycosides show appetite suppressant activities following oral ingestion. This study describes the validation of a bioanalytical method for the quantification of one of the steroid glycosides, H.g.-12 (～10% (w/w) of the extract), in mouse, rat, rabbit and human plasma. The method utilises a liquid–liquid extraction with methyl-tert-butyl ether followed by chromatographic separation on a 2.1 × 50 mm C₁₈ Genesis high performance liquid chromatography (HPLC) column and detection on a triple quadrupole mass spectrometer. Detection of H.g.-12 and its stable isotope internal standards is performed using positive TurboIonspray? ionisation in multiple reaction monitoring mode. The validation procedure demonstrated assay sensitivity, linearity, accuracy, precision and selectivity over the calibration range of 0.5–150 ng/mL in human plasma (500 μL sample volume), 1.0–100 ng/mL in rat and rabbit plasma (150 μL sample volume) and 1.0–250 ng/mL in mouse plasma (150 μL sample volume) with good recoveries (≥77%). H.g.-12 was stable in plasma for ≥6 months at ?20 °C, for up to 4 h at ambient temperature (ca 22 °C) and after 3 freeze–thaw cycles. Plasma extracts were stable for up to 24 h at ambient temperature.     

Xenopus laevis oocytes expressing human P-glycoprotein: Probing trans- and cis-inhibitory effects on [3H]vinblastine and [3H]digoxin efflux

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 °C. [³H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K_m of 145.5 ± 25.4 μM. [³H]Vinblastine (5.6 ± 0.3 μM) and [³H]digoxin (1.0 ± 0.1 μM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [³H]vinblastine and [³H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [³H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug–drug and/or drug–inhibitor interactions.