When the brain structure of interest is clearly displayed, the image should be fixed and zoomed in two- to three-fold for further measurements [11]. The examination is performed at axial scanning planes through the midbrain and the thalami [11] and [12]. The mesencephalic brainstem can be depicted as a butterfly shaped structure of low echogenicity surrounded by the highly echogenic basal cisterns. The echogenicity of the ipsilateral SN, red nucleus (RN) and the BR could be evaluated (Fig. 1). The BR is usually seen as a highly echogenic continuous line with an echogenicity that IWR-1 mouse is identical to that of the RN [13]. Echogenicity of BR is rated semiquantitatively, using either a 3-point (grade

1: raphe invisible; grade 2: slightly echogenic or interrupted BR; grade 3: high echogenicity

identical to that of RN or basal cisterns) or, preferably, a 2-point (grade 0: invisible, hypoechogenic or interrupted BR; grade 1: highly echogenic BR as PD 332991 a continuous line) grading system [13]. It is important to scan the subject investigated from both sides, as the bone window may vary allowing sufficient visualization of the BR only if both sides are considered. Therefore, if the BR can be depicted as continuous line from one side, it is rated as a normal (grade 1) – that is, hyperechogenic, non-interrupted continuous line. Changes in raphe echogenicity reflect changes in tissue impedance and point towards an alteration of the brainstem microarchitecture which could be due to a shift in tissue cell density, a change in interstitial matrix composition, or an alteration of fiber tract integrity [5] and [14]. Various anatomical, physiological, and biochemical findings underline the importance of the basal

limbic system in the pathogenesis of affective disorders, and compelling evidence suggests that the nuclei, fiber tracts, and neurotransmitter systems associated with the basal limbic system are involved in the pathogenesis of primary depression and depression associated with some neurodegenerative diseases such as PD [15] and [16]. The change of acoustic impedance, which is recorded by TCS as reduced Idoxuridine BR echogenicity, might be the result of microstructual changes, gliosis and disruption of fiber tract integrity [14]. Numerous evidence from neuroimaging, biochemical and animal studies implicates basal limbic system and raphe nuclei involvement in the pathogenesis of the mood disorders, particularly depression. Typical ultrasound marker that can be of value in the diagnosis and differential diagnosis of depression is the low echogenicity or interrupted BR. Raphe hypoechogenicity is a common finding in 50–70% of patients with unipolar depression [2] and [17] and is associated with responsivity to serotonin-reuptake inhibitors (SSRI) [18]. In a pioneer study, echogenicity of the BR was examined by TCS in 20 patients with unipolar depression and 20 healthy adult controls.

e. to very short echo times 2τ). The PROJECT (Periodic Refocusing Of J Evolution by Coherence Transfer) approach uses a CPMG sequence with quadrature 90° pulses inserted in the middle of each double spin echo, and is based on the so-called

perfect echo [32] and [33]. The extra 90° pulses refocus J-evolution, for arbitrary τ in AX spin systems and for all spin systems if τJ ≪ 1. If diffusion weighting is added, for example by including field gradient pulses in each echo as in the PROJECTED (PROJECT Extended to DOSY) sequences of Fig. 2, then spin echo DOSY spectra may be obtained free of both exchange effects and J modulation if τk, τJ ≪ 1, where k is the exchange rate constant. The DOSY spectrum of Fig. 1a was acquired for Trametinib purchase a mixture of flavone and catechin. At first sight there appear to be two impurities present. In fact these signals check details are simply the flavone hydroxyl resonances, but their diffusion coefficients are increased by exchange with the small amount of (protio-) water present in the sample. As noted above, such signals are typically better dispersed than backbone proton signals, but serve only to confuse in the spectrum of Fig. 1a. If exchange effects are suppressed using the PROJECTED sequence (Fig.

2, first and last gradient pulses omitted, no 45° pulse), however, the assignment of the signals becomes obvious: hydroxyl and backbone signals alike align correctly in the diffusion domain, as shown in Fig. 1b. Of course the effect is not

limited to exchanging OH signals (which can, if appropriate, be suppressed by addition of D2O), but is general (and extends to magnetization exchange through the Overhauser effect). The use of PROJECT-based DOSY experiments is not limited to cases where exchange is a problem; if, as in small and medium-sized molecules, T2 is not too short, they can be significantly more sensitive than their STE counterparts, because the SE retains the full signal while the STE discards half. However, as there is signal loss due to T2 during the SE delay, for signals with a short T2 a compromise between degree of diffusion weighting and signal-to-noise ratio may be required. Other important examples of applications for PROJECTED include T2-filtered DOSY and convection compensation. T2-filtration is Fenbendazole commonly used where broad signals, for example from polymers or proteins, obscure signals of interest, and is typically implemented in STE-based DOSY pulse sequences by adding CPMG sequences either before [34], or after [35] the STE element. With PROJECTED, diffusion encoding and T2-filtering can be performed simultaneously, minimising signal losses, sample heating and J-modulation. Convection compensation is a particularly attractive application. In STE-based DOSY experiments, convection compensation is normally achieved using a double stimulated echo (DSTE) [22], with a fourfold loss in signal compared to a SE experiment.

6d). Cytotoxicity assays are useful to indicate the ability of a compound to cause cell death as a consequence of damage to one or more cellular functions (Weyermann et al., 2005). Among the cytotoxicity assays for the detection of cell viability following exposure to chemicals, the LDH leakage assay, a protein assay (SRB), the NR assay and the MTT assay are the most commonly employed (Fotakis and Timbrell, 2006). The different mechanisms which result in cell GSK-3 cancer death may influence the sensitivity of the cytotoxicity assay used. The variation of incubation times may be an alternative to reduce these differences of sensitivity. It was reported that the LDH assay gives satisfactory

responses using cell membrane damaging agents, but results obtained with this assay are sometimes misleading if the toxic agent only influences intracellular activities. Such alternatives as the MTT and NR assays can be used to indicate some of these internal damages. The cytotoxic activity in B16F10 cells of G8 and G12 used in this study was investigated in previous studies from our laboratory by measuring the cellular metabolic activity by the MTT method (Locatelli

et al., 2009). In this work, the temporal evaluation of this this website effect revealed that the cytotoxicity of gallates G8 and G12, evaluated by the MTT assay, occurred after 24 h of incubation with the gallates’ amounts corresponding to the IC50 (Fig. 1a and b). In a more comprehensive evaluation using different cell viability assays, it

was observed that G8 and G12 promoted more significant changes in lysosomal activity and cell membrane permeability than interference in mitochondrial activity. Our study revealed an IC50 value about six times lower with the NR assay or three times lower with the LDH assay than with the MTT and SRB assays (Fig. 2a and b). This difference can be due to the fact that the plasma membrane, which is the first site exposed to the compounds was attacked easily when compared to mitochondria an internal drug target. This effect can also be related with cell death in the late apoptotic process in vitro, when membrane integrity is impaired. Cyclin-dependent kinase 3 Concerning if the gallates enter or not into the cell or if they are able to interact with lipid membranes, in a study comparing the activity of dodecyl gallate with its precursor compound, gallic acid, that lacks the hydrophobic alkyl moiety, it was suggested that the dodecyl group allow to partition into cells and organelle lipophilic membranes ( Kubo et al., 2002). The authors proposed that the head and tail structure of hydrophilic pyrogallol moiety of gallates bind to the hydrophilic portion of the membrane surface; in the meantime the alkyl length moiety interferes with the hydrophobic interior surfaces of the membrane.

, 1992 and Bader and Wrbitzky, 2006). Based on these extrapolated CEV concentrations, the proportions above the reference value were used. For the non-smokers, the reference value is clearly defined in the literature, i.e. 10 pmol/g globin (Kraus et al., 2009). In contrast,

for smokers, the reference value in the general population is less unequivocal (Kraus et al., 2009). For this study, an extrapolated CEV concentration of 200 pmol/g globin was used as cut-off for the smokers. CEV concentrations above the reference value of 10 pmol/g globin were observed in 37.3% of the non-smokers in the EZ as compared to 11.5% of the ‘Controls’ outside the EZ. The 95th percentiles were 635 and 22 pmol/g globin, respectively (Table 4). In contrast, in the smokers (Table 5), the proportion selleck inhibitor exceeding Selleck Pictilisib the reference value of 200 pmol/g globin was higher in the ‘Controls’ outside the EZ (68.4%) as compared to the EZ (40.0%). The 95th percentiles and the maxima, however, were higher in the EZ than in the ‘Controls’. To verify whether the CEV concentrations above the reference values in the group of ‘Controls’ outside the EZ could be explained by misclassification by residential address and/or by occupational exposure to ACN, an additional interview was done in all three

non-smokers and in 8 of the 13 smokers. Two of the non-smokers and 5 of the 8 smokers reported they had been in the EZ at the time of or in the days following the train accident.

None of the persons was working in the production of polymers, …. occupationally. Five smokers did not participate in the additional interview and thus kept Thymidine kinase the classification ‘outside the EZ’ as based on their residential address. After correction for localisation as obtained by the additional interview (Table 4), one (4.2%, CEV concentration of 16 pmol/g globin) of the remaining non-smokers outside the EZ had CEV concentrations above the reference value in contrast with 37.3% in the EZ (Chi-square test, P value = 0.003). In the remaining smokers outside the EZ ( Table 5), 57.1% kept CEV concentrations above the reference value, as compared to 40.0% in the smokers of the EZ (Chi-square test, P value = 0.394). In zone 1 (EZ1) and zone 2 (EZ2), 50.0% and 35.0% of the non-smokers (Table 4) had CEV values above the reference level of 10 pmol/g globin, respectively (Chi-square test, P value = 0.310). In zone 1 (EZ1), the concentrations did not exceed a remarkably low maximum of 65 pmol/g globin, the 95th percentile being 36 pmol/g globin. In contrast, in zone 2 (EZ2), the highest CEV concentrations of the whole local population were observed.

Average normalized spectra obtained for roasted coffee and the adulterants spent coffee grounds, roasted coffee husks, roasted corn and roasted barley are shown in Fig. 1. Sharp significant absorption bands can be clearly seen at 2924–2925 and 2852 cm−1, together with absorptions at 1715–1745 and 760 cm−1 in the spectra corresponding

to roasted coffee, corn and barley. Such bands suggest the presence of compounds containing PF-562271 long linear aliphatic chains and, with the presence of absorption bands above 3000 cm−1, are indicative of the likelihood of some of them being unsaturated. Hence, these bands can be partly assigned to unsaturated and saturated lipids present in coffee, corn and barley oils, which are known not to undergo changes during roasting (Reis et al., 2013). Similar bands have also been previously identified in spectra of roasted (Craig et al., 2012a; Kemsley et al., 1995; Reis et al., 2013; Wang & Lim, 2012) and crude coffee samples (Craig et al., 2011, 2012b) and also in spectra of caffeinated beverages such as coffee, tea and soft drinks (Paradkar & Irudayaraj, 2002). In this last specific study, the second band (∼2852 cm−1) was attributed to stretching of

C–H bonds of methyl (–CH3) group in the caffeine molecule and employed in predictive models for quantitative analysis of caffeine. Notice that the second band is less Tacrolimus ic50 evident in the spectra for barley and corn in comparison to the others. Corn and barley do not contain any caffeine, whereas coffee husks are known to have caffeine (∼1 g/100 g dry basis) content similar to those of coffee beans (Fan, Soccol, Pandey, Vandenberghe, & Soccol, 2006). In FTIR studies on corn and corn flour, two bands have also been identified at 2927–2925 and 2855 cm−1 and respectively attributed to asymmetric and symmetric C–H stretching in lipids (Cremer & Kaletunç, 2003; Greene, Gordon, Jackson, & Bennett, 1992). Given the lipids content is not expected to vary during roasting of corn (or barley), the peaks assignment to C–H stretching in lipids might still be valid. The reported

amounts of lipids (Gouvea, Torres, Regorafenib mouse Franca, Oliveira, & Oliveira, 2009; Moreau, 2002; Oliveira, Franca, Mendonça, & Barros-Junior, 2006; Osman, Abd El Gelil, El-Noamany, & Dawood, 2000) of coffee husks (1.5–3 g/100 g) and of barley (1.9–2.87 g/100 g) are lower than those of coffee beans (12–16 g/100 g) and of corn kernels (3–5 g/100 g). Therefore, such bands may be affected by both caffeine and lipids levels in the case of coffee, and are most likely primarily associated to caffeine in the case of coffee husks and only to lipids in the cases of roasted corn, roasted barley and spent coffee. Recall that the majority of the caffeine present in coffee is extracted during soluble coffee production whereas the lipid fraction is partially extracted, hence, leading to spent coffee grounds virtually devoid of caffeine but still containing some lipids.

curvisetus and Rhizosolenia delicatulaP. T. Cleve, 1900 at beach 6, and the green algae Oocystis borgei J. Snow 1903 at beach 9. The Chlorophyta contribution to the total phytoplankton was the highest in winter. During spring, the seasonal cycle of phytoplankton abundance was characterized by a peak corresponding to diatom blooms dominated by Nitzschia spp. (46.60%) and S. costatum (16.70%). The total phytoplankton abundance varied between 0.17 × 104 cells l−1 (beach 10) and

15.61 × 104 cells l−1 (beach 5) with a seasonal Selleckchem Erlotinib mean value of 3.96 × 104 ± 5.29 × 104 cells l−1. Diatoms dominated the phytoplankton at all the sampling beaches. The development of Chlorophyta and Cyanophyta cell abundance also reached a maximum in spring. Spatial fluctuation in spring showed wide variation in abundance and dominant species. Nitzschia GSK-3 signaling pathway palea, N. sigma (Kützing) W. Smith, 1853, and to a lesser extent Pseudo-nitzschia seriata (P. T. Cleve, 1883) H. Peragallo in H. & M. Peragallo, 1900, which formed the bulk of the phytoplankton abundance at beach 5. The dominant species in the phytoplankton community were S. trochoidea (a dinoflagellate) and Dactyliosolen fragilissimus (Bergon) Hasle apud G. R. Hasle & Syvertsen, 1996, Striatella unipunctata (Lyngbye) C. Agardh, 1830 (diatoms) at beach 1, L. flabellata at beach 2, A. granulata at

beach 3, S. costatum at beach 4, Chaetoceros socialis H. S. Lauder, 1864 at beaches 6 and 8, Pseudosolenia calcar-avis (Schultze) Sundström, 1986 at beach 7, and A. minutum at beaches 9 and 10, the last-mentioned species sharing the community with several diatom species such as N. palea, Pleurosigma sp. and Rhizosolenia delicatula P. T. Cleve, 1900. During summer, the seasonal mean value of total phytoplankton cell abundance was 4.32 × 103 ± 2.69 × 103 cells l−1. The total abundance varied between 0.33 × 104 cells l−1 (beach 1) and 1.11 × 104 cells l−1 (beach 7). The dominant group was Bacillariophyta at all beaches except for beach 4 in which Pyrrophyta was predominant. C. closterium formed the main bulk of phytoplankton abundance at beach 7. Nitzschia microcephala

Grunow in Cleve & Möller, 1878 was predominant at beach 1, R. stolterfothii at beach 2, A. granulata at beaches 3 and 10, the Resveratrol last-mentioned species being co-dominant with the green algae Crucigeniella rectangularis (Nägeli) Komárek, 1974, C. marina and Pandorina sp. at beach 10. A. granulata was the dominant species at beaches 4, 5, 8, and 9, and was co-dominant with C. marina at beach 4, C. closterium at beaches 5, 6 and 8; A. granulata and S. trochoidea were the dominant species at beach 9. In general, the overall average cell abundance was 1.45 × 104 cells l−1, and the highest cell abundance of phytoplankton was observed in spring due to the high Bacillariophyta abundance at beach 5. The statistical relationships between the composition of phytoplankton and the physicochemical environment variables at the different sites were analysed.

, 1998 and Ohta et al., 2009) or the PPARγ-agonist and human-specific hepatotoxicant troglitazone at physiologically relevant concentrations (Loi et al., 1999 and Yokoi, 2010). The cytotoxicity of the tested drugs was find more assessed by the release of LDH from cells into the media. The amount of viable and metabolically active cells upon drug-treatment was determined via quantitation of ATP (see Materials and methods section). Rat and human 3D liver cells were treated for 1 to 15 days and 2D hepatocyte monolayers for 2 days with

increasing concentrations of fenofibrate (including the human Cmax of 12.4 μM (Table 1, (Vlase et al., 2010)). Fenofibrate induced dose- and time-dependent toxicity in rat 3D liver model (Fig. 4A) as detected by increased LDH

release and decreased ATP levels upon 15 days treatment. Fenofibrate- induced cytotoxicity in the rat 3D liver model was apparent starting from day 8 of chronic drug treatment. However almost no cytotoxicity was detected after 1–2 days of fenofibrate treatment neither in the rat 3D liver model nor in the rat 2D hepatocyte monolayer cultures (Fig. 4A). Fenofibrate decreased the ATP levels by about 20% in rat 2D hepatocyte monolayers after 2 days of treatment and by 80% in rat 3D liver cells after 15 days of treatment (Fig. 4A). In human 3D liver cells and 2D hepatocytes monolayers, fenofibrate did not induce dose- or time-dependent toxicity (Fig. 4A). Next, we treated rat and human 3D liver cells for 8 days and 2D hepatocyte monolayer cultures for 2 days with increasing concentrations of troglitazone buy MG-132 (including human Cmax of 6.3 μM (Table 1), (Loi et al., 1999)) and measured cytotoxicity and viability of the cells. Troglitazone caused a dose- and time-dependent increase in LDH release and a decrease in ATP levels

in human 3D liver cells but less toxicity was detected in human 2D hepatocyte monolayers (Fig. 4B). Troglitazone induced strong LDH release at physiological Rebamipide relevant concentrations already after 1 day of treatment of human 3D liver cells. The LDH release was more pronounced after 1 day than after 8 days treatment at 50–100 μM, indicating an early effect of this drug on human hepatic cells. In contrast to the results obtained in human 3D liver cells, rat 3D liver cultures did not show marked cytotoxicity and no pronounced decrease in cell viability when incubated with similar concentrations of troglitazone. These results are in line with the data from previous studies demonstrating no troglitazone toxicity in rats at physiological relevant concentrations (Fig. 4B, (Li et al., 2002)). However, troglitazone induced strong increase in cytotoxicity and decrease in cell viability in rat 2D hepatocytes after 2 days of treatment (Fig. 4B). We investigated whether human 3D liver models would detect toxicity induced by drugs known to be hepatotoxic in the clinic (Kaplowitz, 2005).

” …” And I’ve seen it done too many times, where the families actually, everybody gets separated over one old boy dying” (#W2-1) and: “They going to start fighting, don’t put them through it, cut ‘em off. Nope. That’s the reason you prepare your family. You make your wishes before, a will or something” (#H1-3). Participants who favored letting others decide expressed three different reasons: trusting others to decide while giving them guidance (“Authorizers”); Selleckchem Epigenetic inhibitor having complete faith in others that

they would know what to do (“Absolute Trusters”); and letting others decide as a way of avoiding decision-making, i.e., letting others decide by default and without giving them guidance (“Avoiders”), see Fig. 2. Authorizers trusted other family members to decide for them while providing them with general-value guidance or discussing a few hypothetical scenarios: “I think it’s very important that whoever this person is well acquainted with your particular

situation. And you’ve already talked with them and explained or discussed some of the issues involved to the extent that they know. They’re not just guessing, they know what’s going to be best for you.” (#A2-2), and “I prepared them for it and I told them already and at least, I haven’t written click here it down, but I got a will and everything else. But, tell them, I don’t want to, I don’t want no machines. When I can’t GPX6 go to the bathroom, you might as well just pull the plug” (#H1-4). If they did not anticipate any family conflict, they felt less need for writing decisions down, e.g., in the form of a living will: “first my wife

and secondly would be my daughter. Oh, they know. We’ve discussed it. We have discussed it. Many, many times and they both are together on that. They are not one of them pulling one way, the other one the other way. They both agree on everything I want,” (#H2-1), or: “Uh, I don’t have anything in writing, because when I ask my sisters that’s just like printing it in gold, stacking it in gold. They’re going to do it (#A2-1). Absolute Trusters, because of a close relationship, completely believed in their surrogates’ ability to make the ‘right’ decisions for them and were agreeable to and accepting of any such decisions they might make for them. “I tell my daughter to take care of this. … because I know her very good. Because I just know, that’s it, the only answer,” (#H1-4). “I tell my wife to speak. My wife, she got the same power I got. [..

After 6 days of feeding on these diets, control and infected bees were collected for RNA and hemolymph extraction. Ovary status-dependent on the supplied diet was checked in the non-infected groups fed on syrup, beebread or royal jelly. In a parallel experiment, six groups of 40 bees from three

colonies (two groups per colony) were collected and separately maintained Gefitinib nmr in screened wooden cages during 9 days in the same conditions of temperature and RH described above. During this period all bee groups were continuously fed with beebread collected from a single hive. To one group from each colony it was given water (control group), and the other group from the same colony (experimental group) received water containing S. marcescens (105 bact/ml). Therefore, each pair of experimental/control groups was taken from the same colony.

Water (pure and contaminated) was given ad libitum. After 9 days the bees were dissected and their ovaries were classified as non-activated if ovarioles were slender, without growing follicles, (comparable to the stage 1 categorized by Pirk et al., 2010), or were considered activated if containing growing follicles (comparable to stages 2–4) or fully-developed follicles (comparable to stage 5). After hemolymph collection (item 2.4), total RNA was extracted from dissected abdomens (integument and adhered fat body), using TRIzol reagent (Invitrogen). Samples containing selleck products 1 μg of total RNA were treated with DNAse (Promega) and used for reverse transcription with Superscript II (Invitrogen) and Oligo (dT)12–18 (Invitrogen). Aliquots of cDNA were subjected to quantitative (real-time) RT-PCR and semi-quantitative RT-PCR. Gene expression levels in abdomens of Thiamine-diphosphate kinase bees fed different diets, infected or not with S. marcescens, were analyzed using the 7500 Real Time PCR System (Applied Biosystems). Amplification was carried out with a 20 μl reaction volume, containing 10 μl of SYBR® Green Master Mix 2× (Applied Biosystems),

1 μl of cDNA (diluted 10×), 7.4 μl of water and 0.8 μl (8 pmol) of each gene-specific primer. The working genes (GenBank accession numbers is underlined) and respective primer sequences were: vg (AJ517411) forward 5′-GCA GAA TAC ATG GAC GGT GT-3′ and reverse 5′-GAA CAG TCT TCG GAA GCT TG-3′; vgr (GB16571) forward: 5′-ACC TTA CGA CAT TGC CCT-3′ and reverse: 5′-TGT GAT TTT CGG TCC AAG CCC-3′; apoLp-II/I (GB11059) forward 5′-AGC GAA GAG GAT CGC AGA TA-3′ and reverse 5′-AAC CCT TCG TTC CTC CTT TC-3′; apoLpr (XP_395858.3) forward 5′-GGT CGT TCA TGT ATA TCA TCC-3′ and reverse 5′-CGG ACA AGC ACA ACT AAG-3′; apoLp-III (ABY82793) forward 5′-TCT GAC AAA GCT GCG AAA TC-3′ and reverse 5′-AGT TGC GGC AGT TTG AAG TT-3′; and hex 70a (ABQ59246) forward 5′-GCT GGT ATC TGA ATC ACG ATT-3′ and reverse 5′-CAC GAT AAT CCG GCA AAT CG-3′. The PCR conditions were 50 °C for 2 min, and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and the temperature is 60 °C for 1 min.

Immunoreactive bands were visualized using a chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, UK), followed by autoradiography. β-actin was used as the loading control. Densitometry of various analyte proteins and their respective loading controls from the same blot was performed using Image J 1.43 (NIH) software. Relative optical density was calculated by

dividing the densitometry of analyte(s) protein with the respective loading control. The levels of BPDE-DNA adducts were detected by immunohistochemical staining for BPDE-DNA adducts in formalin-fixed, paraffin embedded 5 μm tissue sections as described previously [14]. Sections were incubated with anti-BPDE antibody (1:30

dilution). Detection was done using Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine http://www.selleckchem.com/products/Bortezomib.html (DAB) was employed as the chromogenic substrate, and slides were counterstained with Mayer’s haematoxylin. Images were captured with Zeiss Microscope (Imager Z1) to which an Axiocam MRc5 digital EGFR inhibitor camera was attached. Quantitative analysis of the images (magnification X 400) was performed by IHC profiler [15], which is an open source plug-in for the quantitative evaluation and automated scoring of immunohistochemistry images of tissue samples. This modified digital image analysis is based on protocols adopted earlier [16]. IHC photomicrographs were used for developing semi-automated analysis protocol, namely IHC profiler [15]. As a first step, a color de-convolution plug-in was used to un-mix the pure DAB and haematoxylin stained areas that left a complimentary image. The pixel intensities of separated DAB images range from 0 to 255. Value 0 represents the darkest shade whereas 255 represents the lightest shade of the DAB brown color in the image. To select the DAB-stained (brown) nuclei, the threshold feature of the Image J 1.43 (NIH) software was used. Further to assign an automated percentage of pure DAB staining patterns in the nucleus, a macro was developed and plugged in the Image J 1.43 (NIH) software to obtain an automated counting

of the pixel wise percentage contribution of high, medium and low positive pixels/intensity in an image i.e. the number of pixels of a specific intensity value GPX6 vs. their respective intensity zone. For measurement of BPDE-DNA adducts [similar areas of tissue sections (mm2) and number of cells (∼800 cells/section/animal)], total intensity (%) [of nuclei containing percentage of high, medium and low intensity] was analyzed within different treatment groups. However, apoptosis was measured in terms of total apoptotic nuclei intensity as well as percentage of apoptotic positive and negative cells in similar areas of tissue sections (mm2) and number of cells (∼800 cells/section/animal) in different treatment groups.