, 1995, Colasante et al , 1996 and Ueda et al , 2006), probably a

, 1995, Colasante et al., 1996 and Ueda et al., 2006), probably as the result of a non-specific cell membrane disturbing action ( Kreger, 1991 and Chen et al., 1997). Albeit few pharmacological activities were described for Sp-CTx

(hemolysis and vascular tonus modulation) ( Andrich et al., 2010), the neutralisation of the local inflammatory and cardiovascular effects of crude S. plumieri venom by SFAV-treatment offers evidence that this scorpionfish cytolysin may possess other pharmacological actions. However, further studies are necessary to assess such potential features of this cytolysin. The results obtained demonstrated that the stonefish antivenom evoked an immune cross-reactive response with scorpionfish venom. SFAV is efficient learn more in neutralising the most prominent toxic effects of scorpionfish venom. find more This is in accordance with the hypothesis that venomous fish belonging to

different genera or inhabiting different regions may share venom compounds with similar antigenic properties (Church and Hodgson, 2002b). This resemblance may rely on the fact that piscine venoms have evolved for a same defensive purpose and possess similar multifunctional cytolytic toxins (Saunders, 1960, Russell, 1965, Kreger, 1991, Church and Hodgson, 2002b and Andrich et al., 2010). Finally, the study of such venoms and/or toxins may be useful for developing new and more specific antivenoms (or even antibodies) targeting specifically the fish venoms membrane-disturbing toxins and helping in alleviating the major symptoms of scorpionfish envenomation. This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico – 477514/06-5), DAAD-CAPES (Probral 250/06), INCTTOX (Instituto Nacional de Ciência e Tecnologia em Toxinas) and FACITEC (Fundo de Apoio à Ciência e Tecnologia do Município de Vitória). We would like

to express our gratitude to Dr. Michael Richardson for revising this manuscript. The authors are indebted to M.L.B. Goze for capturing the fishes. “
“Monocrotaline (MCT), a pyrrolizidine alkaloid phytotoxin, has well-documented hepatic and cardiopulmonary toxicity for animals, including ruminants and humans (Mclean, 1970, Mattocks, 1986, Huxtable, 1989, Souza et al., Vasopressin Receptor 1997, Schultze and Roth, 1998, Stegelmeier et al., 1999, Nobre et al., 2004 and Nobre et al., 2005). This compound is frequently ingested accidentally because of food grain contamination or intentionally in the form of herbal medicine preparations (Huxtable, 1989). It has been reported that its toxicity depends on cytochrome P-450 mediated bioactivation to the reactive pyrrolic metabolite dehydromonocrotaline (DHM) (Butler et al., 1970, Lafranconi and Huxtable, 1984, Roth and Reindel, 1990, Wilson et al., 1992, Pan et al., 1993 and Schultze and Roth, 1998). This metabolite, despite having a half-life of only a few seconds in aqueous media, is a powerful alkylating agent that binds to DNA and proteins (Petry et al., 1984, Hincks et al.

Then, 20 μL of this suspension was injected into the HPLC (Shimad

Then, 20 μL of this suspension was injected into the HPLC (Shimadzu, C18 reverse

phase and UV detection at 280 nm) and eluted with a gradient of acetonitrile and 0.175 (g/100 mL) orthophosphoric acid ( Makkar et al., 1997). We use phorbol-12-myristate 13-acetate (Sigma) as standard. The soluble protein and reducing sugar contents were determined according to the colorimetric methods described by Bradford (1976) and Miller (1959). For this analysis, 10 g of the crushed mushrooms was placed in Erlenmeyer flasks (125 mL) containing 25 mL sodium citrate buffer AG-014699 clinical trial (0.05 mol/L and pH 4.8). These flasks were kept in a shaker for 30 min at 150 rpm, and the extracts were filtered using Millipore membranes (Cavallazzi et al., 2004). This experiment was conducted using a completely randomized design with 5 replicates. The data were subjected to analysis

of variance, and the averages were compared by Tukey’s test (p < 0.05) using Saeg software (version 9.1, Federal University of Viçosa). The tannin concentrations Selumetinib datasheet observed in the jatropha seed cake (Fig. 1) were similar to those found in the fruit peel of J. curcas ( Makkar, Aderibigbe, & Becker, 1998). The greatest concentration of this compound was observed in the substrate with coffee husk and eucalypt bark ( Fig. 1). This may have been due to the presence of tannins in the eucalypt bark ( Vázquez, González-Alvarez, Santos, Freire, & Antorrena, 2009) and in the coffee husk ( Barcelos, Paiva, Pérez, Santos, & Cardoso, 2001). The thermal treatment Methamphetamine of the substrates decreased the tannin concentration by 46%

(Fig. 1). This result was similar to that observed in vegetables after cooking or autoclaving at 121 °C and 128 °C for different periods of time (Rehman & Shah, 2005). Regardless of the substrate, tannin degradation by P. ostreatus Plo 6 increased as a function of the incubation time, and the highest rate was observed between 15 and 30 d in the substrate with coffee husk and eucalypt bark ( Fig. 1). The high degradation rate of this compound was also observed in Pleurotus sp. cultivated in coffee husk for 60 d ( Fan et al., 2006) and this tannin degradation may be related to tannase activity (tannin acyl hydrolase, EC The activity of this enzyme in the polyphenols degradation has been reported in Aspergillus and Penicillium ( Batra & Saxena, 2005). Thus, P. ostreatus can degrade the tannins in J. curcas seed cake and the other tested substrates. The acid phytic content in the jatropha seed cake (Fig. 2A) was lower than the percentage this acid found in the seed of J. curcas by Makkar et al. (1998). This result shows that a percentage this acid was in the oil. Although phytic acid is considered to be heat-stable (Deshpande & Damodaran, 1990), the sterilization of the substrates decreased in 20% the content of phytic acid (Fig. 2A).

4C) Differential loading of the antigen on the resin could resul

4C). Differential loading of the antigen on the resin could result

in different OAg chain conformations and a different exposure of the epitopes and/or determine a different interaction with the corresponding antibodies. The present study describes the successful coupling of OAg from S. Typhimurium to NHS-Sepharose in order to produce an affinity matrix that is capable of purifying specific antibodies from polyclonal human serum. The method can be applied to OAg from different bacteria and it does not modify OAg chain epitopes. Columns were filled with different OAg–ADH preparations with consistent results and columns were used at least 10 times with no deleterious effect on recovery of antibodies. This process could potentially be adapted for the purification of antibodies against other bacterial polysaccharides, as well as for the large scale purification of antibodies against Salmonella OAg. Y 27632 As such, the method will be useful for the investigation of the protective capacity of OAg antibodies with different fine specificities in the context of

immunity to NTS, both in HIV-infected African adults and in individuals immunised with OAg-based vaccines. We find more thank Simona Rondini for the extraction of OAg from S. Typhimurium D23580. We are grateful to Robert Heyderman and the Malawi-Liverpool-Wellcome Trust Clinical Research Programme for S. Typhimurium D23580. We thank Simona Rondini, Yunshan Goh and Adam Cunningham for helpful discussions. This work was supported by a PhD studentship from the Medical Research Council, UK (to CMO’S) and a Clinical Research Fellowship from GlaxoSmithKline (to CAM). The authors 6-phosphogluconolactonase declare that they have no conflict of interest. “
“Assessing cytokine profiles in small

tissue biopsies presents a significant technical challenge, particularly the quantification of multiple cytokines when some are present at low concentrations. Multiplex methods using Luminex technology may offer an attractive solution. However these are often developed using soluble materials such as sera or cell culture supernatants spiked with recombinant cytokines and standard protocols are not available for tissue samples. Luminex assays use multiple sets of polystyrene or paramagnetic beads or ‘microspheres’ — see Vignali (2000) and Houser (2012). Each set is fluorescently colour-coded to be identifiable on a dedicated flow cytometer or other platform and pre-coated with antibody to capture a specific cytokine or other analyte, around which a sandwich immunoassay is built. Different bead sets can be combined to enable simultaneous measurement of multiple cytokine concentrations in a single sample against standard curve preparations. These assays require substantially less sample than traditional enzyme-linked immunosorbent assays (ELISAs) – typically 25–50 μL for multiple analytes compared with 200 μL for a single analyte – yet may offer similar sensitivity to Luminex (Vignali, 2000, Biagini et al.

The reconciliation with the maximum

The reconciliation with the maximum

selleck inhibitor parsimony gene tree resulted in eight duplications and 24 extinctions (Fig. 6), while the Bayesian gene tree showed eight duplications and 23 extinctions and maximum likelihood gene tree nine duplications and 29 extinctions. These events of duplication and differentiation of the genes occurred over a period of about 22 million years, the timeframe for the evolution of viperid snakes in the New World (Wüster et al., 2008). The high number of extinctions may be due to the lack of other β-defensin-like genes from the same species as well as from other Bothrops snakes. The evolution of these genes occurred according to the birth-and-death model, as for β-defensin genes and other

multigene families in vertebrates ( Nei and Rooney, 2005) and as suggested for the crotamine and crotasin genes ( Oguiura et al., 2009). We amplified β-defensin-like sequences of several snakes and we noticed that their genes have the same organization as the crotamine and crotasin genes as well other β-defensin-like genes of lizards and fishes. The evolution of genes is dynamic, where not only do substitutions occur but also intron gains and losses (Babenko et al., 2004). Coulombe-Huntington and Majewski (2007) observed a trend toward intron losses in mammals; furthermore, they observed that intron losses occurred more frequently in those smaller than 150 bp. We proposed that the structure of three exons and two introns is a squamate characteristic, because it is found in snakes and lizards, whereas the feature of two exons is characteristic for mammals (Patil et al., 2005) and four exons PLX3397 for birds (Xiao et al., 2004). All β-defensin-like sequences that have been described show a common main gene organization in a particular group of animals, but also one or more sequences Orotic acid with a different structure: our DefbBa01 has only two exons, some in lizards have four exons ( Dalla Valle

et al., 2012), and mammals also have genes with more than two exons ( Patil et al., 2005). In summary, all animals possess two or more gene structures, but with the predominance of one. As the β-defensin-like genes of zebrafish are organized in three exons and two introns (the first in phase 1 and the second in phase 2; Zou et al., 2007), and the ray finned fishes are the basis of the species tree ( Shen et al., 2011), we speculate that the ancestral gene had this gene structure. After the speciation of mammals, the copies with two exons duplicated, and sometime after the speciation of the squamates and birds/turtles/crocodilians group, intron insertions occurred in the β-defensin-like genes, and this different arrangement duplicated more than that with three exons. Only studies of β-defensin-like genes in other animals including turtles and crocodilians and also amphibians and other fishes can further elucidate gene evolution in vertebrates.

The mycelia were recovered from the liquid medium by filtration,

The mycelia were recovered from the liquid medium by filtration, washed with distilled water and dried at 40 °C during 24 h to obtain the yield of biomass. The residual amount of reducing sugars in the filtrates was evaluated by using the dinitrosalicylic method ( Miller, 1959). Fruiting bodies and the mycelia were dried and

milled to a fine powder (40 mesh). The samples (5 g) were extracted by stirring with 100 mL of ethanol 70:30 (in water) at 25 °C and at 130 rpm for 3 h and filtered through Whatman n° 1 paper. Ethanol was chosen because of its abundance and low cost. The extractions were repeated two times. No increases in yield were achieved by further extractions. The combined filtrates were concentrated with a rotary vacuum evaporator at 40 °C to eliminate ethanol, freeze-dried and weighted. The freeze-dried powders www.selleckchem.com/products/SGI-1776.html were stored in freezer until use. The reducing and total carbohydrates of extracts were determined by using the dinitrosalicylic (Miller,

Alpelisib in vivo 1959) and anthrone (Morse, 1947) methods, respectively, and expressed as glucose equivalents. Evaluation of the reducing and non-reducing sugars present in the extracts was also carried out using thin layer chromatography. The samples were spotted onto Silica Gel 60 plates (Merck, Darmstadt, Germany) and developed with butanol–pyridine–water (15:30:20, vol/vol) as the mobile phase. The spots were detected by spraying with 5 g/L KMnO4 in 1 mol equi/L NaOH. Glucose, mannitol and trehalose at 20 g/L were used as standards. Total phenolic compounds were

Selleckchem Fludarabine measured using Lowry reagent (Singleton & Rossi, 1956) and expressed as gallic acid equivalents. The determination of flavonoids was done by means of a colorimetric assay (Zhishen, Mengcheng, & Jianming, 1999) and expressed as catechin equivalents. Compounds bearing free amino groups were measured using the ninhydrin method with l-alanine as standard (Starcher, 2001). Amino acids were evaluated also using paper chromatography (Moffat & Lytle, 1959). The hydroalcoholic extracts from both mycelia and fruiting bodies of A. brasiliensis were fractionated by means of high-performance liquid chromatography (HPLC). The HPLC system (Shimadzu, Japan) consisted of a system controller SCL-10AVP, two pumps, model LC10ADVP, a column oven, model CTO-10AVP, and a UV–Vis detector, model SPD-10AVP. A reversed-phase C18 CLC-ODS column (5 μm; 250 × 6 mm i.d.; Shimadzu) protected with a GHRC-ODS pre-column (5 μm; 10 × 4 mm i.d.; Shimadzu) was used. The absorbance of each sample solution was measured at 280 nm. The mobile phase was distilled water:glacial acetic acid (99.9:0.1) (solvent A) and acetonitrile:glacial acetic acid (99.9:0.1) (solvent B). The gradient was 0 min, 92:8 (A:B); 0–2 min, 90:10 (A:B); 2–27 min, 70:30 (A:B); 27–50 min, 10:90 (A:B); 50–60 min, 0:100 (A:B); 60–63 min, 92:8 (A:B). Run time was 63 min using a flow rate of 1 mL/min ( Kim et al., 2008).

All authors contributed to the conception and design of the study

All authors contributed to the conception and design of the study, selection of patients, laboratory analysis, data analysis and interpretation, and drafting of the manuscript. All authors contributed to and read

and approved the final manuscript. IM and PK are guarantors of the paper. This work was supported by a grant from The Wellcome Trust (07664/Z/05/Z, PK) and TC is a recipient of an Imperial College London MB/PhD fellowship. None declared. The study protocol was approved by the UK NHS National Research Ethics Service (COREC reference 05/Q0410/93). “
“Laboratory diagnosis of acute Leptospira infection in endemic settings is problematic as there is a paucity of simple, inexpensive, well characterised assays that are diagnostically informative. The serological ‘gold standard’ is the microscopic agglutination see more test (MAT), which requires paired specimens Selleck LDK378 and considerable technical resources

and training and, in some cases, is not useful for acute patient management. 1 Simple IgM antibody detection-based ELISAs are marketed as being accurate for the diagnosis of Leptospira infection, however their accuracy is dependent on the background immunity of the local population and the number of days of illness. 2 Here we report the evaluation of a commercial ELISA for the detection of Leptospira IgM antibodies among adults with fever in the leptospirosis-endemic setting of the Lao People’s Democratic Republic (Laos) to determine (i) its utility for diagnosis of acute leptospirosis and (ii) a locally appropriate diagnostic cut-off. Human serum was collected as part of a previously described study2 following informed consent as part of a study to determine the causes of unexplained fever in patients presenting at Mahosot Hospital (Vientiane, Laos) between November 2001 and October 2003.3

Admission (n = 184) and convalescent (n = 151) sera were collected from 184 patients (total samples, n = 335) and were stored at –20 °C until tested. Ethical approval was granted by the Ethical Review Committee of the Faculty of Medical Sciences, National University of Laos, Vientiane, Laos. Miconazole A commercial ELISA (Standard Diagnostics, Yongin-si, South Korea) for the detection of IgM antibodies against Leptospira spp. was performed according to the manufacturer’s instructions at Mahidol University–Oxford Tropical Medicine Research Unit in Bangkok, Thailand. An optical density (OD) of ≥0.75 was defined as positive according to the manufacturer’s method. Reference serology methodologies have been described elsewhere. 2 The MAT for Leptospira antibodies was performed by the WHO/FAO/OIE Collaborating Centres for Reference & Research on Leptospirosis at KIT Biomedical Research (Amsterdam, The Netherlands) and in Brisbane (Australia).

For those ports near estuaries (e g Shanghai, New York, Hamburg,

For those ports near estuaries (e.g. Shanghai, New York, Hamburg, etc.), the change in density may be significant, this assumption is clearly not appropriate. In also trying to assess the influence of proposed cleaning technologies (ozone, UV, centrifugal separation), which are applied in transit, the density contrast

between re-injected (cleaner) water and ballast water can be neglected. However, if ballast water is cleaned by heat, the treated water is lighter than the original water. The new element of the paper involves development of a robust multizone model GPCR & G Protein inhibitor for ballast water flushing and a detailed comparison against experimental results. To understand the influence of outlet arrangements and compartment configurations on the flushing efficiency, we examine the temporal and spatial fluid exchange experimentally. The paper is structured as follows. In Section 2, we describe how we set up the mathematical model and the diagnostic tools used to interpret the results which are discussed in the context of simplified geometries; in Section 3, we apply the model on ballast tanks with different compartment configurations and outlet arrangements; in Section 4, we present the experimental

setup and methodology; in Section 5, we analyse the experimental results and compare with the model prediction; in Section 6, we conclude. The water used to flush a ballast tank is typically pumped into the tank at a rate Q~1m3/s. GKT137831 As we move farther away from the inlet nozzle, the mean flow decays quite quickly. It is important to estimate typical values as these variables determine the type of modelling approach and the validity of the experimental analogue described later. The ballast tanks in a large ship are characterised by a typical length L  =50 m, width W  h=20 m of the base and W  v=0.5 m of the vertical Tenofovir purchase region, height H  h=2 m of the horizontal region and H  v=25 m of the vertical region, and the nominal diameter of the inlet nozzle D  =0.5 m. The mean flow velocity through the nozzle is Un=4Q/(πD2)~5m/s. The flow in a ballast

tank is characterised by the Reynolds number Re=UL/νRe=UL/ν, where ν   is the kinematic viscosity of water, ν=10−6m2/s. We start using an assessment of the typical velocity and length scales in a ballast tank, where U   and L   are the characteristic velocity and lengthscale of the flow, respectively. The inlet nozzle Reynolds number is Ren=UnD/ν~106Ren=UnD/ν~106. Based on the horizontal section, Uh≥Q/(0.5πWhHh)~10−2m/s, Reh=UhHh/ν~104Reh=UhHh/ν~104. Up the riser section, Uv=Q/(LWv)~10−2m/s, so Rev=UvWv/ν~104Rev=UvWv/ν~104. Thus the flow in a ballast tank is inertially dominant so that Re is high and the flow is turbulent. The purpose of this model is to quantify how flushing efficiency varies within a tank.

Resultados epidemiológicos semelhantes relativos à incidência anu

Resultados epidemiológicos semelhantes relativos à incidência anual da DACD em doentes hospitalizados foram apresentados em Espanha, com um aumento da incidência anual de 3,9 para 12,2 casos por 10 000 internamentos, entre 1999 e 2007 8. Neste último estudo, o aumento da incidência anual da DACD correlacionou-se com o aumento da proporção de doentes internados a quem foram prescritos antibióticos Forskolin in vitro 8. No presente

número do GE, no artigo intitulado «Diarreia associada ao Clostridium difficile – casuística de 9 anos», Dinis Silva J et al. apresentam uma análise retrospetiva de 37 casos de DACD num hospital distrital, diagnosticados entre 2000 e 2008. Este estudo obriga a refletir sobre as potenciais causas subjacentes ao aumento da incidência desta infeção documentada nos últimos anos. Salienta-se a grande variabilidade da incidência anual da DACD no período estudado: 2/10 000 internamentos em 2000 versus 16/10 000 internamentos em 2008. Este aumento exponencial da incidência no último

ano incluído no estudo é equiparável ao aumento da incidência apresentado por Vieira AM et al. num hospital central português 7. Na análise dos fatores Angiogenesis inhibitor de risco conhecidos de DACD entre diferentes períodos do estudo, os autores salientam 2 dados interessantes: 1 – os antibióticos carbapenemes estiveram mais vezes implicados nos casos de DACD no ano 2008 comparativamente a 2000-2007 (6/16 versus 1/21, respetivamente, p = 0,01); e 2 – a utilização de inibidores da bomba de protões foi proporcionalmente superior nos casos de DACD no ano 2008, comparativamente

a 2000-2007 (11/16 versus 6/21, respetivamente, p = 0,02). Estes dados levam a refletir sobre a potencial influência dos padrões de prescrição de antibióticos e de inibidores da bomba de protões no aumento da incidência da DACD. Ao mesmo tempo que assistimos a uma crescente e preocupante utilização de carbapenemes na rotina hospitalar, em particular nas instituições com elevadas taxas de resistência entre as bactérias Gram negativas 9, surgem relatos de que os carbapenemes poderão associar-se Erastin a um maior risco de DACD comparativamente aos antibióticos que mais se têm associado à infeção por C. difficile nos estudos prévios (penicilinas, cefalosporinas, clindamicina e fluoroquinolonas) 10 and 11. Num estudo retrospetivo recente de grande dimensão, que procurou identificar fatores de risco de DACD após terapêutica antibiótica de infeções pós-operatórias, apenas a utilização de carbapenemes teve influência na incidência de DACD, correspondendo a um aumento do risco de 1,7-vezes comparativamente ao uso de outros antibióticos 10.

If fc > 2 and p < 0 05, assign “Inc” (Increased) If fc < 0 5 and

If fc > 2 and p < 0.05, assign “Inc” (Increased). If fc < 0.5 and p < 0.05, assign “Dec” (Decreased). Otherwise, assign “NC” (Not Changed). 1. When a classifier for increased liver weight was built: Discretization

thresholds for gene expressions combined with fold changes and statistical test (e.g. student’s t-test) have often been applied in microarray data analysis and is reported to be better than p-value alone [22]. In general, numerical parameters obtained in toxicity studies are judged to be increased or decreased, based essentially on statistical comparison with contemporary controls and, if available, additionally on historical data [23]. In this study, we discretized click here liver weights based only on statistical tests, as no historical data was available. Before proceeding to CBA, gene expressions discretized selleckchem as “NC” in each group were discarded from the data, because we were interested only in genes with increased or decreased expressions. We then analyzed the data with CBA, with discretized gene expressions as non-class items and discretized liver weights as class labels. We used the lda function in the MASS library of R. R‘s lda function is implemented based on Rao’s LDA [24] and [25], also known as Fisher-Rao LDA,

which generalized Fisher’s LDA [26] to multiple classes. Prior to the LDA analysis, the data was preprocessed as described in the CBA section, except that gene expressions were not discretized. Before proceeding RVX-208 to LDA, the feature selection step was conducted to reduce the number of genes, because classical LDA requires the total scatter matrix to be nonsingular, while the matrix can be singular when the sample size (149) does not exceed the number of features (genes) (more than 30,000) [27], and tends to overfit and become less interpretable in the presence of many irrelevant and/or redundant features [28].

Based on the previous reports on microarray data analysis [29] and [30], we selected only the genes that were up-regulated (fc > 2 and p < 0.05) or down-regulated (fc < 0.5 and p < 0.05) in the groups with increased or decreased liver weight when compared to the not-increased or not-decreased groups, respectively. To compare predictive performances of CBA and LDA, we conducted 10-fold cross validation [31] for each methods with the total of 149 records(compounds), and evaluated sensitivity, specificity, and accuracy averaged over 10 validations. These parameters are defined as follows [32]. Sensitivity: True Positive/(True Positive + False Negative) Specificity: True Negative/(True Negative + False Positive) Accuracy: (True Positive + True Negative)/Total Full-size table Table options View in workspace Download as CSV 10-fold cross validation, or more generally k-fold cross validation, is one of the standard methods for evaluating predictive performances of classifiers.

In a second experiment, using the same strain and S9, reference s

In a second experiment, using the same strain and S9, reference sample 2R4F again gave the highest revertant yield, but there was no clear

concentration-related increase in mutagenicity for any PM. Two further experiments confirmed weak concentration-related increases in revertants for all of the PARP inhibitors clinical trials PMs, with reference sample 2R4F giving the clearest response. The mutagenic potencies of the extracts in TA1537 were generally lower than they were in TA100, and showed some variation between experiments. In one of the three experiments with a concentration-related increase in revertants, conducted with TA1537, W862 and W863 were significantly less mutagenic than W860, W861 and W864; and W863 and W864 also exhibited significantly lower potencies than W861 in two experiments. In conclusion, there were no qualitative differences between PMs in any strains. The PMs were also the same in terms of S9 dependence. Quantitatively, PMs with 80% BT tobacco Galunisertib were less mutagenic than the other PMs in strain TA98 with S9 activation. All PMs induced

dose-related increases in cytotoxicity, and also induced genotoxicity with and without S9 and at the different treatment times. PMs increased the frequency of micronucleated binucleate cells by more than 3-fold. In terms of dose and %MnBn/μg NFDPM, the 20 h treatment without S9 was more sensitive than the 3 h treatments. At 20 h without S9, W862 induced fewer micronuclei than W860 and W861 in both experiments

(Fig. 2). This was statistically significant in both experiments for W860 and in one experiment for W861 (Table 6). At 3 h ± S9, W862 induced fewer micronuclei than W861, in two experiments (Table 6). The assay’s resolving power was limited by Metformin in vivo relatively large variability within and between experiments, non-linear responses and >50% cytotoxicity at the higher doses in the 3 h treatments. This may have contributed to the inconsistent differences observed between PMs. Concentrations of test and reference PMs were selected in order to provide as many points as possible lying on the linear part of concentration–response curves, and to provide as many concentrations as possible that were common to each PM treatment, whilst allowing treatment up to toxicity limiting dose levels. In some cases the highest concentration levels were not selected for plating to determine viability and TFT resistance, or were excluded from analysis, due to excessive toxicity (based on cell count data). Statistically significant increases in mutation frequency (MF) of 3- to 4-fold were observed with each of the PMs, on each experimental occasion and with each of the treatment conditions employed (e.g. Fig. 3). In terms of dose and MF/μg NFDPM, the 20 h treatment without S9 was the most sensitive, and the 3 h treatment with S9 was the least sensitive.