Extracts from the flowering plant, lisianthus, were used for comparison. The extracts showed differential effects in the chemical assays, suggesting that closely related structures have different affinities to scavenge different reactive species. Integration of anthocyanins to an AVI led to more sustained radical scavenging activity as compared with the free anthocyanin. All but the red lettuce extract could reduce endogenous DNA damage in HT-29 colon cancer cells. However, while extracts from purple sweetpotato skin and flesh, Maori potato and pansies, protected cells against subsequent challenge by hydrogen peroxide at 0°C, red cabbage extracts were pro-oxidant, while other extracts had no effect. When the peroxide challenge was at 37°C, all of the extracts appeared pro-oxidant. Maori potato extract, consistently the weakest antioxidant in all the chemical assays, was more effective in the Comet assays. These results highlight the dangers of generalising to potential health benefits, based solely on identification of high anthocyanic content in plants, results of a single antioxidant assay and traditional approaches to structure activity relationships. Subsequent studies might usefully consider complex mixtures and a battery of assays.DNA damage by reactive species is associated with susceptibility to chronic human degenerative disorders. Anthocyanins are naturally occurring antioxidants, that may prevent or reverse such damage. There is considerable interest in anthocyanic food plants as good dietary sources, with the potential for reducing susceptibility to chronic disease. While structure-activity relationships have provided guidelines on molecular structure in relation to free hydroxyl-radical scavenging, this may not cover the situation in food plants where the anthocyanins are part of a complex mixture, and may be part of complex structures, including anthocyanic vacuolar inclusions (AVIs). Additionally, new analytical methods have revealed new structures in previously-studied materials. We have compared the antioxidant activities of extracts from six anthocyanin-rich edible plants  using three chemical assays , and the  However, the uncontrolled increase of ROS is strongly associated with the etiology and pathophysiology of a number of chronic human diseases such as inflammation, viral infections, cancers, autoimmune, neurodegenerative, cardiovascular and digestive system disorders, as well as premature aging. High levels of ROS have been reported to alter the structure and function of cell membranes, adversely affecting cell function . The humIn addition to their aesthetic qualities in food, various examples of major plant pigments, including polyphenols such as anthocyanins, have been shown to have metabolic and nutritional benefit, partly through their antioxidant effects . AnthocyCirculatory and cardiovascular diseases may be reduced by the antioxidant actions of anthocyanins, as demonstrated by the increased resistance to hydrogen peroxide-induced ROS generation of red blood cells (RBC) treated with these compounds. Many anthocyanins appear to show superior antioxidant properties to other related polyphenols  as well Anthocyanic vacuolar inclusions (AVIs) contain high numbers of anthocyanin molecules, stacked onto a protein matrix . They arIf an anthocyanin-rich food is to have significant influence on health, it becomes important to consider levels in commonly eaten foods that could realistically be incorporated into the human diet. Some good examples are summarised in Lettuce and cabbage are common dietary items for which red variants have good anthocyanin levels, and are commonly available in the food supply . BlueberViola × wittrockiana  and Eustoma grandiflorum (lisianthus), the latter of which is known to have anthocyanins in an AVI [An additional difficulty in determining human health implications of anthocyanin-rich foods, is that the anthocyanins are not found as isolated compounds, but rather as a component of often complex mixtures. Possible pro-oxidant actions at certain concentrations also need to be considered . In thisn an AVI . Three d2.2.1.Anthocyanin contents of 1 g of fresh material as extracted into 10 mL of acidified methanol are compared in This figure illustrates that 99N1/222 flesh, pansies, lisianthus, red cabbage and red lettuce had similar levels of anthocyanins. However, as compared with extracts from these five plants, the extract from Maori potato flesh had an approximately 6-fold lower anthocyanin content, that from the blueberry extracts showed approximately two thirds of the anthocyanin content, while the 99N1/222 skin extract had an anthocyanin content more than 50% higher.2.2.50) and are displayed in 50 values, except for the Maori potato, which had about a 13-fold higher IC50 value compared to 99N1/222 skin extract, which had the lowest IC50  incubation at room temperature .After 15 minutes, the TRAP activity of the free anthocyanic solution was higher as compared with extracted AVIs, and no activity was seen in the extract from the non-pigmented lisianthus. Radical scavenging by the free anthocyanins was saturated after 15 minutes, with no further increase after 24 hours. In contrast, intact AVIs continued to scavenge the ABTS radical, showing markedly superior total antioxidant activity compared to free anthocyanins after 24 hours. Unfortunately, seasonal differences in the varieties of lisianthus available have made it impossible to extract sufficient quantities of AVIs to repeat this observation.2.4.Unlike the previous assays, the ORAC assay measures the area under the curve of the time course of extinction of the luminescent compound R-PE. Higher values, therefore, indicate better antioxidant activity. The ORAC assay reveals more variation in the antioxidant activities of each extract .® standard (which has comparable antioxidant activity to 1 mM ascorbic acid). Red cabbage extract showed slightly lower antioxidant activity than the standard, while Maori potato extract was again the least effective antioxidant, about 6-fold lower than the pansy extract, which had the highest ORAC value.Six of the eight extracts, namely those from the 99N1/222 skin, flesh, pansies, lisianthus, blueberries and red lettuce extracts showed a higher ORAC value than the 1 mM Trolox2.5.Three variations of the Comet assay were performed. In the first, cells were incubated for three days with or without the previously identified maximum non-toxic dose of the anthocyanic plant extracts. The cells were then assayed for DNA damage without further treatment (untreated group). In the other two groups, after three days of incubation with or without the extracts, the cells were resuspended in PBS and challenged with hydrogen peroxide for 15 minutes at either 0 ºC or 37 ºC . For each of the treatment groups, the average tail extent moments of 100 comets for the control cells (no extract) plus the cells incubated with the various extracts were calculated and are described below.2.5.1.In the group that did not receive hydrogen peroxide treatment, all the extracts cause a reduction in average tail extent moment or tail length relative to the control . The taiThe majority of the extracts were able to provide a significant reduction (p≤0.05) in constitutive DNA damage, with only the red lettuce (p=0.098) extract unable to do so. The 99N1/222 skin, which had the best overall protection, was also significantly better than all the other extracts in terms of reducing background DNA damage (p≤0.01). 99N1/222 flesh and red cabbage extract was significantly better than the bottom ranked two extracts, Maori potato  and red lettuce , while pansy and blueberry extracts were significantly better than red lettuce . The remaining extracts, from lisianthus, Maori potato and red lettuce, showed no significant differences in ability to protect against endogenous DNA damage (p>0.05).2.5.2.In addition to the protection from endogenous free radicals examined in the previous section, the effects of the various extracts on subsequent challenge by exogenous hydrogen peroxide was also studied. By performing these assays at 0 ºC, enzymatic effects could be eliminated. Therefore, any antioxidant effects observed in response to the extracts would be due to direct scavenging of hydrogen peroxide entering the cells, or by prior lowering of the cell redox state so that it can absorb more ROS before damage occurred.In the group that received hydrogen peroxide treatment at 0 ºC, the control had increased DNA damage over the controls in the non-peroxide treated group. However, not all the extracts were able to cause a reduction in average tail extent moment relative to the control .Relative to the untreated cells, hydrogen peroxide increased DNA damage in the controls (p<0.0001) .Four of the extracts, 99N1/222 skin and flesh, Maori potato and pansies, significantly reduced hydrogen peroxide damage by approximately half (p<0.05). Lisianthus, red lettuce and blueberry extracts had no significant effect (p>0.05) and red cabbage extract actually caused an increase in DNA damage to about twice that of the controls (p=0.002).2.5.3.In the group that received hydrogen peroxide treatment at 37 ºC, the control cells showed the least amount of DNA damage .Unexpectedly, none of the extracts were able to reduce DNA damage induced by hydrogen peroxide at 37 ºC to below the control .With the exception of 99N1/222 skin extract (p=0.159), all the extracts produced significantly more DNA damage relative to the control (p<0.05). Pansy, 99N1/222 flesh, Maori potato and lisianthus all produced DNA damage at 37 ºC that was indistinguishable from their respective values at 0 ºC (p>0.05). The red lettuce extract, while still inducing more DNA damage relative to the control (p=0.002), had less damage at 37 ºC than at 0 ºC (p=0.010). DNA damage at 37 ºC and 0 ºC with the red cabbage extract did not significantly alter (p=0.508). The blueberry extract, which showed neither protective nor deleterious effects relative to the control at 0 ºC (p=0.367), exhibited twice the DNA damage at 37 ºC than at 0 ºC (p=0.003).2.5.4.The degree of hydrogen peroxide stimulated DNA damage in the presence of each of the extracts at both 0 ºC and 37 ºC is compared in The p-values show that DNA damage in the controls and red lettuce has significantly reduced at 37 ºC, but that damage induced by 99N1/222 skin and flesh, Maori potato, pansy, lisianthus and red cabbage extracts have stayed the same at both temperatures. In contrast, the damage induced by blueberries has significantly increased at 37 ºC in comparison to the 0 ºC treatment.2.6.The three chemical assays evaluated measure primary antioxidant activity of the anthocyanic extracts, while the Comet assay can also measure secondary effects. However, the chemical assays are quick and simple methods of measuring potential antioxidant activity, whereas utilising oxidation of cellular components can be time consuming to perform and not practical where large numbers of samples are involvedThe common feature of the three chemical assays is their direct measurement of free radical scavenging efficiency of the extracts. The DPPH assay is the quickest and easiest assay to perform, but it diverges from biological conditions the most, using an artificial DPPH radical and methanol as the solvent . This meThe ORAC assay measures the degree and length of time the extracts take to inhibit the action of an oxidizing agent. It therefore takes into account the kinetics of the reaction, unlike the other two assays, as well as being performed at a physiological pH and producing a biologically relevant radical, the peroxyl radical . Since aFar more biologically relevant is the Comet assay, which visualizes DNA damage in single cells arising from the exposure to various combinations of antioxidants and ROS ,29.. CelThe initial set of COMET assay results measure constitutive DNA damage in cells without exposure to an exogenous source of free radicals. They demonstrated that most of the extracts could protect the DNA from damage by endogenous free radicals generated by the cells during their 3 day incubation period. 99N1/222 skin offered the best protection of all of the extracts tested, while the red lettuce extract did not offer any significant protection relative to the controls. The remaining extracts, while showing significant improvement from the control, were in the middle ground in terms of their antioxidant capacities when comparing between extracts.Of the extracts that were able to offer some protection against hydrogen peroxide challenge, none were able to completely protect, indicated by the fact that there was still significantly more damage than in cells exposed to the respective extracts but not hydrogen peroxide. A possible explanation for this is that the Comet assay is performed in PBS, giving the extracts the opportunity to diffuse out of the cells, thus reducing intracellular protective effects. One interesting observation is that the Maori potato extracts, which were consistently the least protective antioxidant in the chemical assays, appear to be the most protective against ROS induced DNA damage at 0 ºC . PerhapsAlso interesting is the observation that the second best antioxidant in the untreated group, red cabbage extract, now appears to be pro-oxidant. This indicates that, while red cabbage extract does not induce DNA damage directly, it contains other compounds which can enhance the deleterious effects of exogenous ROS in a non-enzymatic manner. Metal ions, for example, are able to synergistically increase the effects of ROS .Whereas performing the hydrogen peroxide challenge at 0 ºC showed direct scavenging of exogenous ROS by the extracts, performing the hydrogen peroxide challenge at 37 ºC also allowed enzymatic effects to be taken into account. Surprisingly the average tail extent moment seen in the 37 ºC hydrogen peroxide challenged controls was reduced back to around the same value as the non-hydrogen peroxide challenged controls. This may suggest that protective antioxidant enzymes such as catalase and glutathione peroxidase, which convert hydrogen peroxide into water and oxygen, as well as DNA repair enzymes, have sufficient protective capabilities to reduce ROS induced damage to background damage. To somePrevious structure-activity relationships have suggested that antioxidant capacity varies considerably according to the pattern of substitution of the anthocyanin molecule, the presence of acyl groups, and the nature and positions of glycosyl groups . HoweverIn our studies, the TRAP assay revealed that AVIs and equimolar free anthocyanins have very similar antioxidant capacities in the short term, with the antioxidant potential of the AVIs only slightly less than that of the free anthocyanins. However, observation of the plate used in the TRAP assay after 24 hours showed that the AVIs, but not the free anthocyanins, had continued to scavenge the ABTS radical. These results show that although AVIs are slower in scavenging free radicals, they have greater total antioxidant activity based on an equimolar anthocyanin concentration. This may be due to the highly organised structure of the AVIs allowing free radicals to be delocalised across the many aromatic rings of the packed anthocyanins.Although we have not done in vivo studies here, other researchers have reported that consumption of anthocyanins or anthocyanin-rich diets leads to increased serum antioxidant potential in both experimental animals and human subjects ,36. Addi3.3.1.Urenika (purple skin/purple flesh) cultivars were grown in an Auckland University glasshouse and were the kind gift of Dr Kevin Gould, School of Biological Sciences, University of Auckland, Auckland. All other plant materials were field grown. 99N1/222 sweetpotato was the kind gift of Dr Steve Lewthwaite, New Zealand Institute for Crop and Food Research Limited, Mt. Albert, Auckland. Pansies were purchased from Palmers Garden Centre, Glen Eden, Auckland, Blueberries from First Choice Limited, Wiri, Manukau, while red cabbage and red lettuce were both from Foodtown, Greenlane, Auckland. Lisianthus was purchased from In Vogue Blooms, Auckland, New Zealand. The lisianthus used was a deep purple-flowered variety Wakamurasaki, and materials extracted from the even deeper purple throat. During the experiments, fully mature and healthy material was harvested and then thoroughly washed at least three times with 1 liter of tap water containing three drops of Tween-20 prior to a final wash with MilliQ water.3.2.λmax values in the 500–550 nm waveband as measured using a Hitachi dual beam spectrophotometer. The phenolic composition of some of the extracts was determined after 2D-paper chromatography (2D-PC) as described by [Approximately 10 mL of extract per sample was prepared in the required solvent (7% acetic acid in methanol for the methanolic extract or Milli-Q water for the aqueous extract) at a concentration of 10% fresh weight per volume. The homogenizer  tip was washed with the extract solution before use and the plant material was homogenized in a 50 mL conical tube with 5 mL of the extract solution until completely macerated. The homogenized solution was filtered using a Büchi B-169 vacuum system  through a 60 mL F glass filter (Kontes) into a 250 mL vacuum flask , both pre-washed in the extract solution. The remaining required volume of extract solution was used to wash both the 50 ml conical tube and the glass filter, before the wash solution was filtered through and combined with the initial filtrate. Extracts were freeze dried and stored at −20 ºC. The methanolic extract is designated anthocyanin-rich extract, or ARE. Anthocyanin levels in the extracts were estimated as Aribed by .3.3.50 values were calculated as the volume of extract corresponding to the absorbance midway between the highest absorbance on the plate (extract solution and DPPH only) and the lowest absorbance value on the plate (the highest concentration of the ascorbic acid standard).The methanolic extract was used for these experiments, serially diluted into the extract solution in 96-well plates in 50 μL volumes. One hundred and fifty μL of 100 μM DPPH  in methanol was added to all the wells and the plates were left to stand in the dark for 30 minutes. Absorbance values were read at 515 nm using a Spectra MAX Plus plate reader  and the Softmax Pro 2.4 software. Duplicate absorbencies were averaged and plotted against extract volume. IC3.4.50 values were calculated as above.The buffer reaction mixture was freshly made for each experiment with 5 mL 750 μM ABTS , 10 mL 10 mM AAPH  dihydrochloride) stock solutions and 35 mL 50 mM acetate buffer, pH 4.3. The 50 mM acetate buffer, pH 4.3 was made up of 100 mM acetic acid and 100 mM sodium acetate. The reaction mixture, which is light sensitive, was incubated at 45°C for 60 minutes, then returned to room temperature. Serial dilutions of each aqueous extract were made as for the DPPH assay. Two hundred μL of the reaction mixture was added to all the wells, plates were left to stand in the dark for 15 minutes, and absorbencies were read at 734 nm at 25°C using a Spectra MAX Plus plate reader  and the Softmax Pro 2.4 software. IC3.5.0 = fluorescence at 0 minutesThis was performed as previously described . The meti minutesfi= fluorescence at k = dilution factor (1000)ORAC values were then calculated from these S values:3.6.90), and this concentration was used for subsequent experiments. Cells were incubated at 37 °C for three days, in the presence of the IC90 of the appropriate ARE. They were PBS washed, the supernatant discarded and the pellet re-suspended in 2 mL supplemented media and kept on ice while cells were being counted. Cell numbers were determined using a Coulter Cell Counter . Cells were centrifuged  at 800 rpm for 10 minutes and the supplemented media supernatant discarded before the cells were resuspended in the appropriate volume of PBS to give 1 × 106 cells/mL.Human colon cancer (HT-29) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum, penicillin (100 unit/ml), and streptomycin (100 mg/ml). Cells were incubated at 37°C in a 5% CO2 incubator, and subcultured before reaching 90% confluency at 2–3 day intervals. Preliminary growth inhibition assays established the concentration of ARE to reduce cell growth by 10% over a three day incubation period  while the third tube received 20 μM hydrogen peroxide at 37 °C (in a water bath). After 15 minutes incubation, the tubes were centrifuged at 1,000 rpm for 3 minutes and the supernatants were discarded.Slides were prepared treated and electrophoresed as described by Singh . DNA dam3.7.Kolmogorov-Smirnov normality analysis showed that, of the three chemical assays, only the ORAC assay produced normally distributed data (p>0.02). This lack of normality in the DPPH (p=0.003) and TRAP (p<0.001) assay data sets make regression analysis inappropriate to determine whether correlations exist between the various assays. Therefore, the nonparametric method of Spearman rank order correlation was used to determine relationships between data from the chemical assays.The averages, standard deviations and standard errors of the 100 comets scored for each extract and treatment applied were calculated using Microsoft Excel 2000 . This information was plotted on bar graphs using the SigmaPlot 2000 software  with the standard errors being represented by the error bars. Frequency distributions for each extract and treatment applied were also plotted. P-values were calculated with the t-test function from SigmaPlot 2000  to compare between the extracts plus the control for each separate treatment. The t-test function was also used to determine the p-values comparing between the 0 ºC and 37 ºC treatment groups. P-values less than 0.05 were taken to indicate a statistically significant difference. Kolmogorov-Smirnov normality tests and Spearman rank order correlations of the three chemical assays and the three Comet treatment groups were performed using SigmaStat v2.03 .4.Anthocyanins are very complex, and the behaviour of even apparently closely related structures may be different. The primary result of the present studies is that what one assay suggests as their antioxidant potential may be quite different to the results suggested by another assay. Furthermore, the way in which the anthocyanin presents in the cell may be especially important. At least in lisianthus petals, the AVIs in the central vacuoles are derived from the aggregation of anthocyanins into insoluble structures that are similar to membranous networks in appearance. This structural aggregation may lead to significantly different antioxidant properties from those of the isolated molecules. In addition, anthocyanins are typically present in complex mixtures and may interact with one another and with other polyphenols in establishing their biological properties.


The associations were examined by univariate analysis while multivariate logistic regression was used to examine the determinants of odour annoyance. The results showed that odour annoyance was significantly correlated to modelled pollutants at the individual  and census tract  levels. The exposure-response relationships show that residents of Sarnia react to very low pollution concentrations levels even if they are within the Ontario ambient air quality criteria. The study found that exposure to high NO2 and SO2 concentrations, gender, and perception of health effects were significant determinants of individual odour annoyance reporting. The observed association between odour annoyance and modelled ambient pollution suggest that individual and census tract level annoyance scores may serve as proxies for air quality in exposed communities because they capture the within area spatial variability of pollution. However, questionnaire-based odour annoyance scores need to be validated longitudinally and across different scales if they are to be adopted for use at the national level.This study aimed at establishing the relationship between annoyance scores and modelled air pollution in “Chemical Valley”, Sarnia, Ontario (Canada). Annoyance scores were taken from a community health survey (N = 774); and respondents’ exposure to nitrogen dioxide (NO Most epidemiological studies that assess health effects of pollution use fixed monitoring sites and different modelling techniques , but adequate information and data are not always available . For exaRecently, land use regression (LUR) modelling has been proposed as an alternative approach to address some of the limitations by assessing the intra-urban spatial variability of ambient air pollutants in urban and industrial settings. LUR modelling captures localized variations in ambient air pollution more effectively and economically than some of the conventional approaches discussed above . In addiet al. [2) pollution levels and annoyance scores at individual levels. In Sweden, Forsberg et al. [2) and annoyance scores. When comparing self-reported traffic intensity to modelled air pollution from traffic in three birth cohorts from three countries: the Netherlands; Germany; and Sweden, Heinrich et al. [2 modelled estimates. In addition, while examining the relationship between publicly available air quality data and public perception of air quality in London, UK, Williams and Bird [Annoyance caused by air pollution has been suggested as an indicator for ambient air pollution exposure ,10. Thiset al.  found nog et al.  reportedh et al.  found weand Bird  reportedet al. [2 concentration at home, but also found that smoking, workplace dust exposure, and respiratory symptoms were significant predictors of individual annoyance score. While Forsberg et al. [2, they did report a high correlation between NO2 concentration and annoyance related to air pollution and traffic exhaust fumes. Furthermore, Jacquemin et al. [2 concentration levels in 20 cities from 10 European countries. They concluded that annoyance scores could be a useful measure of perceived outdoor air quality. Smith et al. [2 concentrations in three Swedish cities . In Oslo, Norway, Piro et al. [On the other hand, while examining whether a questionnaire-based indicator (annoyance) of ambient air pollution can be a useful proxy for assessing the within-area variability of air quality in Switzerland, Oglesby et al.  reportedg et al.  reportedn et al.  reportedh et al.  found thh et al.  reportedo et al.  found th2 and SO2 at individual and census tract levels; b) to examine the individual determinants of odour annoyance caused by industrial pollution; and c) to establish exposure–response relationship between NO2 and SO2 exposure and odour annoyance.This study extends the emerging literature on the relationship between odour annoyance and air pollution in Sarnia “Chemical Valley”, Ontario (Canada), a sentinel high exposure environment; with the following specific objectives: a) to determine the correlations between odour annoyance score and modelled NO2.et al. [et al. [et al. [This study is informed by risk perception and odour annoyance literature –21. In get al.  found th [et al.  reported [et al.  found th [et al. . These dScientists assume they have more objective understanding of risk due to their rigorous experimental studies, epidemiological surveys, and probabilistic risk analysis; but the lay persons’ understanding of risk is based on misperceptions or misunderstandings of the objective  risk . Howeveret al. [et al. [et al. found that the frequency of reporting annoyance was high among women and people with respiratory illnesses like asthma. These discussions above provide a background for interpreting the relationship between modelled pollution and annoyance, and the determinants of annoyance within the context of a highly exposed environment.Odours from industrial sources, such as the petrochemical plants in Sarnia, have been shown to considerably impact general health and well-being by affecting both the physiological and psychosocial status of people ,25. Suchet al.  argued tet al. , p. 412  [et al.  identifi [et al. ,26,30, a [et al. ,26. For 3.3.1.2 and a population of 71,419 [Sarnia is located in Southwestern Ontario, Canada, and has an approximate land area of 165 kmf 71,419 . The Cit3.2.2 and SO2 were monitored at 39 locations across the city of Sarnia for two weeks. LUR was utilized to model the intra-urban spatial variability of measured ambient NO2 and SO2 concentrations [As part of a larger community health study aimed at examining the determinants of health in Sarnia, a multi-method approach including qualitative and quantitative approaches were used. Detailed descriptions of these approaches are found elsewhere –34. In btrations . Figure 3.3.3.3.1.2 and SO2 pollutants based on their 6-digit postal codes. As covariates, indoor exposures were measured by asking respondents the number of indoor appliances including air humidifiers, filters and conditioners they have in their dwellings. Residents were also asked whether they were exposed to dust or fumes at home or work.The developed LUR model equations  were useThe influence of five health variables including self-assessed health status, chronic conditions, emotional distress, cardinal, and general health symptoms were examined. Self-reported health status  was asse3.3.2.Study respondents self-assessed their degree of annoyance due to air pollution odours on an 11-point annoyance indication  through the following question: “How much are you annoyed by odours from the chemical plants at your actual home, if you keep the windows open?” Similar scale has been validated in other European studies .3.4.st quartile, moderate: 2nd and 3rd quartiles, and high: 4th quartile). In addition, the continuous individual and census tract mean annoyance scores were correlated and regressed against the individual and census tract mean pollution estimates. In total, there were 20 valid census tracts (out of 24) that had individuals whose mean annoyance scores and pollution estimates were determined. From the total sample, 30 respondents had missing annoyance records, and they were dropped from the analysis .Both univariate and multivariate approaches were used to examine the relationship between annoyance score and the LUR modelled exposures at the individual and census tract levels. The individual level measurements are respondent’s records of annoyance score or estimated exposure while census tract level measurements are mean values of all individual annoyance scores or estimated exposure that fall within each census tract—subdivision of a county. Annoyance scores at the individual levels were related to gender, age, and estimated pollution quartiles  developed by the Ontario Ministry of Environment [2 and SO2 concentrations was “100 ppb”. The correlation coefficients between modelled NO2 and SO2 were 0.49 and 0.65 at individual and census tract levels, respectively. Overall, 34% of respondents reported no annoyance at all; 50% reported little annoyance (1–7); and 16% reported high annoyance (≥8). The overall mean annoyance was 3.22 with a median of 2. The mean LUR modelled pollution concentrations for individuals (N = 774) was 13.82 ± 1.64 ppb for NOironment  for NO2 2 and SO2 pollution concentrations. Female respondents reported comparatively higher mean annoyance scores than their male counter parts. The results also showed a wide range of perception at a given level of ambient air pollution based on age group (The results show that annoyance scores increase with increasing NOge group . When th2 (r = 0.15) and SO2 (r = 0.13) at the individual level compared to the high coefficients of 0.56 and 0.67 at the census tract level for NO2 and SO2, respectively . In model III, the introduction of occupational exposure to dust made significant effect on odour annoyance score reporting with those more exposed at work more likely to report odour annoyance . When sociodemographic variables, general health status , and perception of odours were controlled (Model IV), the odd ratios of reporting high odour annoyance increased for people exposed to high NO2 pollution quartiles. Residents exposed to the 3rd and 4th NO2 concentrations quartiles were more than 3 times more likely to report high annoyance than residents who are exposed to the 1st and 2nd pollution concentrations quartiles. In the final model . With the first order interaction, the final NO2-based model showed an improved model with acceptable goodness of fit (ρ2) of 0.21 [In the multivariate logistic regression analysis, each possible determinant was evaluated one at a time, and a dichotomized odour annoyance rating (annoyance score ≥ 8 versus < 8) of industrial odour disturbances was used as an outcome variable. We examined the influence of modelled NOModel V, , high po of 0.21 . The goo of 0.21 .2 concentrations and high odour annoyance scores remained significant even after we controlled for age and gender . Upon the inclusion of sociodemographic variables, general health status , and perception of odours, the 3rd and 4th SO2 concentrations quartiles gained strength as predictors for high odour annoyance reporting  but remained significant. Individuals who reported more than two cardinal symptoms were more than two times more likely to report high odour annoyance. Respondents who believed that odours would adversely impact their health were more than five times more likely to report high annoyance compared to individuals who were neutral or are in disbelieve that industrial odours will impact their health .The relationship between modelled SO2 concentrations quartiles maintained their influence on high odour annoyance , Cox and Snell R2 (0.17), and Nagelkerke R2 (0.29) [In addition, residents who perceive that odours have not improved in the last five years were 80% more likely to report high annoyance than individuals who believed that odours related to the industrial pollution in their region have improved . When the first order interaction effects were introduced into the model, the high SOModel V, . The int2 (0.29) .2 and SO2 pollutions have significant influence in high odour annoyance reporting even in the context of other covariates. The overall contributory effects of the mediating variables suggest that these variables may be exacerbating the impact of pollution in self-reporting of high odour annoyance.In general, NO2 concentration level, the estimated parameters values , at least 85% of respondents are a little annoyed and more than 47% are highly annoyed by odours in the region (2 (47%) compared to NO2 pollution (37%) at the same pollution concentration levels. At the same pollution levels, about 87 and 85% of Sarnia residents are at least a little annoyed to NO2 and SO2 pollution respectively. It should be noted that there are few or no concentrations for NO2 above 17 ppb and above 12 ppb for SO2 modelled concentrations. The range of 0–50 ppb was used to facilitate comparisons.With the same concentration of SOe region . The res5.2 and SO2 concentrations are below Ontario’s AAQC, a considerable proportion of residents surveyed were annoyed by the odours. The study found a strong association between annoyance scores and the different LUR modelled ambient air pollutants including NO2 and SO2. Consistent with other studies [et al. reported high correlations between annoyance and NO2, they found no significant relationship between annoyance scores and SO2 concentrations in their study context [This study examines the relationship between odour annoyance and modelled pollution, exposure– response associations, and the determinants of annoyance scores in the highly exposed environment of Sarnia. Although modelled NO studies ,16, peop context . When co2 concentration had higher coefficient (r = 0.67) compared to NO2 (r = 0.56) at the census tract level. At the highly annoyed threshold , health variables including self-assessed health status, chronic conditions, emotional distress, cardinal, and general health symptoms showed significant influence on the high annoyance reporting. However, when covariates were introduced, only cardinal symptoms reporting and their interaction with exposure to occupational dust showed significant influence on odour annoyance score reporting. These results signify the importance of covariates in modifying the influence of these health variables on odour annoyance reporting. Further, studies should explore how single health outcomes  rather than using composite health variables  impact odour annoyance reporting. While examining the influence of individual health outcomes on self-reported air pollution problems, Piro et al.  found thet al. [2 and SO2 pollution concentrations in Sarnia [2 were more likely to report higher annoyance scores than when they are exposed to NO2 concentrations. This is partly because SO2 and its reduced compounds  are known to produce pungent smells detectable at very low concentration levels and can directly result in ill-health such as nausea or headache [2 and SO2 pollution concentration, cardinal symptoms reporting, and odour annoyance scores suggesting that odour annoyance is influenced by pollution levels and other covariates. This finding is consistent with Luginaah et al.’s who found that strong odours may result in ill-health  reporting [Explaining the causal linkages between odour annoyance, odour perception, and health outcomes could be problematic. While examining the community reappraisal of perceived health effects of a petroleum refinery in Oakville, Ontario, Luginaah et al.  highlighn Sarnia . When coheadache . In thiseporting . Second,2 and SO2 in Sarnia [2 and SO2 concentrations but were not measured in this study. The use of a 6-digit postal code instead of personal monitoring to develop air pollution estimates might also be another limitation. Nevertheless, the findings make important contributions to the literature.Despite the fact that the models used in this study captured the significant proportion of the spatial variability of NOn Sarnia , there a2 and SO2, and odour annoyance reporting is mediated by odour perception and health outcomes [et al.’s [In general, this study provides strong support for the second mechanism, whereby the relationship between exposure to industrial pollutants including NOoutcomes ,30. Thiset al.’s  who repo2 and SO2 concentration guidelines. Considering the relationship between odours mediated mechanisms and health effects, the results suggest the need to revisit the set guidelines for allowable exposure to pollution to better protect residents. This finding is consistent with Amundsen et al. [The exposure–response relationships indicate that residents of Sarnia are annoyed with pollution concentrations levels that are very low compared to the Ontario 24 hour allowable NOn et al.  who founIn conclusion, questionnaire-based odour annoyance score of ambient air pollution can be a useful proxy for assessing the within-area variability of air quality and measure of perceived ambient exposure and could be used for evaluating the implementation of environmental policies. In fact, as a subjective score of air quality, odour annoyance has been incorporated in the Swedish National Monitoring programs . Odour a


ICl,swell channel in the adult cardiac myocyte.Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive ICl,swell inhibition in control cells decreased time to half-maximal volume , consistent with a role for ICl,swell in volume regulation. MBCD-treated cells showed reduced time to lysis (0.02T) and t0.5,vol (0.64T) compared with controls. The negative inotropic response to swelling  was enhanced by MBCD.Rat ventricular myocytes were exposed to solution of 0.02 tonicity , 0.64T for 10–15 min (swelling), and/or methyl-β-cyclodextrin . MBCD and 0.64T swelling reduced the number of caveolae visualised by electron microscopy by 75 and 50% respectively. MBCD stimulated translocation of caveolin 3 from caveolae-enriched buoyant membrane fractions, but both caveolin 1 and 3 remained in buoyant fractions after swelling. ICl,swell. They illustrate a general principle whereby caveolae as a membrane reserve limit increases in membrane tension during stretch/swelling thereby restricting mechanosensitive channel activation.These data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of  Caveolae are small (50–100 nm) invaginations of the plasma membrane, found in almost all cells of the body. They represent a specialised form of lipid raft, characterised by the presence of the small protein caveolin, which inserts into the inner leaflet of the membrane via a hairpin loop Caveolae have been shown to play a role in a variety of cellular processes including endocytosis, cholesterol homeostasis and signal transduction. Caveolae act as a compartment which brings together elements of the endocytotic machinery, lipid transporters and signal cascade components. Within caveolae, Cav acts as a regulator of protein activity and as a scaffold by interaction via its 20 residue scaffolding domain ICl,swellICl,swell is considered to be acting as a mechanosensor responding to changes in membrane tension, rather than to decreased intracellular osmolarity, because it can also be activated in response to other mechanical stimuli We are interested in the contribution of caveolae to volume regulation during swelling in the cardiac myocyte. In the heart, cell swelling can occur during episodes of ischaemia and reperfusion. During ischaemia, metabolites such as lactate accumulate within the cell causing swelling which is exacerbated on reperfusion, when the hyperosmotic extracellular solution is exchanged for one with normal osmolarity. During swelling, ion channels are activated that regulate cell volume; the main volume-regulatory channel is the swelling activated chloride channel et al.ICl,swell following transfection with Cav 1. This group went on to show that transfection of Cav-expressing endothelial cells with truncated Cav 1 (Δ1–81), which displaces endogenous Cav 1 from the lipid raft membrane fractions, impaired activation of ICl,swell during swelling ICl,swell channel or elements that regulate ICl,swell require caveolae/caveolin. Consistent with caveolae's role as a compartment, it has been suggested that they affect volume regulation by bringing together the ICl,swell channel with tyrosine kinases which regulate channel activity The first direct evidence for a link between caveolae and volume regulation was made by Trouet et al.ICl,swell) by limiting increases in membrane tension for a given change in cell volume. However, the work of Trouet et al.ICl,swell activation.However, caveolae's property as a membrane reserve is also likely to be relevant to processes like volume regulation that depend on changes in membrane tension. The lipid bilayer can only increase in area by around 3% before rupture, so membrane reserves are essential to allow changes in cell volume to occur without irreversible cell damage ICl,swell activation  in the adult ventricular myocyte. Our data are consistent with the hypothesis that caveolae as a membrane reserve attenuate ICl,swell activation by limiting increases in membrane tension during swelling.The aim of the present study was to investigate the effect of swelling on caveolar morphology and density, and to determine the consequences of removing caveolae for processes that depend on n = 16 cells). Apparent surface area increased by 16±1% during this time. The time-course of changes in cell volume represents the sum of increased volume through osmotic swelling and decreased volume through volume-regulatory mechanisms, principally ICl,swell. As volume and apparent surface area measurements are estimates based on a 2-dimensional image of the cell, we consider that it is the time-course of swelling that provides the most sensitive index of processes that modulate volume regulation.We have previously shown that treatment of myocytes with 2 mM MBCD for 1 h at 37°C depletes caveolae of their 2 essential components, cholesterol and Cav 2), which is identical to values reported in adult rat myocardium using transmission EM by Patel et al.2). The average diameter of a caveola is ≈75 nm which (assuming the caveola is a perfect sphere) means that the caveolar surface membrane area will be ≈0.02 µm2. Therefore, open and closed caveolae together contribute around 12% to membrane area in these cells. This is lower than a previous estimate of 22% 2).EM data from control cells  allow usSome mechanical stimuli cause translocation of Cav from caveolae which can have implications for signalling regulated by Cav ICl,swell is unknown, although several candidates have been proposed including C1C-3, P-glycoprotein and phospholemman  in order to estimate the surface and intracellular membrane reserves (which include open and closed populations of caveolae respectively) ICl,swell contributes to volume regulation in the cardiac myocyte, limiting cell swelling. In order to assess the way that caveolae regulate ICl,swell, we compared the effect of ICl,swell inhibition and caveolar disruption with MBCD on the cell volume response to hyposmotic challenge (0.64T). In these experiments, tamoxifen (Tmf) was used to block ICl,swell; Tmf is considered a relatively selective blocker of ICl,swell and at 10 µM fully blocks this current ICl,swell was inhibited with tamoxifen, MBCD-treated cells, and MBCD treated cells in which ICl,swell was inhibited with tamoxifen. There was a trend for the maximal volume achieved during swelling to be higher following tamoxifen and/or MBCD treatment although this was not significant (P>0.05)  effects; both attenuate volume regulation in the cardiac cell.(P>0.05) . However(P>0.05) . TamoxifICl,swell depolarises resting membrane potential and decreases action potential duration ICl,swellICl,swell -induced action potential abbreviation ICl,swell to test how disrupting caveolae affects ICl,swell activation. As Tmf also blocks L type Ca2+ current ICl,swell in experiments involving measurement of cell contractility. In isotonic conditions, DIDS (50 µM) has no effect on action potential configuration ICl,swell and disrupting caveolae on the negative inotropic effect of swelling. ICl,swell inhibition with DIDS reversed the negative inotropic response to swelling, consistent with a role for ICl,swell in this. Although MBCD mimicked the effect of ICl,swell inhibition on the volume response to swelling (potentiation (P<0.05) of the negative inotropic response to swelling at 6 and 10 min after exposure to hypotonic solution relative to controls. This suggests that disrupting caveolae increases activation of ICl,swell.swelling , in cellICl,swell activation in the cardiac myocyte. We have shown that swelling has marked effects on the configuration of caveolae without affecting Cav localisation. Disrupting caveolae has significant effects on volume regulation and contractility in response to swelling, two processes which involve ICl,swell activation. Our data are consistent with the idea that disrupting caveolae removes essential membrane reserves so that cells swell more quickly, thereby potentiating activation of the mechanosensitive ICl,swell channel. A simple model summarising our findings is shown in In this study we have looked at the effect of hyposmotic swelling on the morphology and Cav content of caveolae, and the impact of caveolar disruption on processes that depend on et al.Using electron microscopy we have quantified for the first time the effect of hyposmotic swelling on the morphology and number of caveolae in the isolated cardiac myocyte. We see a 50% reduction in the number of caveolae (identified on the basis of size and shape) after 15 min exposure to hypotonic solution. This change in caveolae number reflects a decrease in the number of closed caveolae for which we cannot visualise a neck which connects with the sarcolemma. No change in the number of open caveolae was recorded. In agreement, Kohl In past decades there has been discussion as to whether closed subplasmalemmal vesicles of a size consistent with caveolae are truly closed caveolae, or whether they in fact represent a population of caveolae sectioned outside the connecting neck region. Early EM studies showed that in smooth, skeletal and cardiac muscle extracellular markers penetrate virtually all subplasmalemmal vesicles of a size consistent with caveolae, suggesting that they are relatively static structures open to the extracellular space We propose that the effect of swelling on reducing closed caveolae without affecting the open configuration underlies a cycle whereby stretch causes flattening of open caveolae in tandem with sarcolemmal incorporation of closed caveolae see . Kohl anDespite effects of swelling on the morphology of caveolae, we saw no translocation of Cav 1 or 3 from the caveolar fraction of the myocytes see . Whilst et al.In the ventricular myocyte, the maximum volume and surface area increase recorded prior to lysis (68 and 24%) are much less than that reported for epithelial cells or fibroblasts (>1000 and 360%) ICl,swell contributes to volume regulation in the cardiac myocyte. Here we have confirmed this by showing that the rate of swelling  in response to 0.64T solution is increased when ICl,swell is inhibited with tamoxifen. Disrupting caveolae with MBCD had an identical effect on the rate of swelling. We know that MBCD causes a translocation of Cav 3 from caveolae and a reduction in the number of identifiable caveolae ICl,swell, in other words, ICl,swell activation relies on caveolae (or the presence of Cav 3). This is in accord with work from Trouet et al.ICl,swell channel with elements necessary for its activation.ICl,swell activation relies on caveolae/Cav 3, then the negative inotropic response observed several minutes after swelling, which has been ascribed to action potential abbreviation secondary to ICl,swell activation enhanced in cells treated with MBCD. These data are not consistent with the hypothesis that ICl,swell relies on caveolae/Cav 3, but suggest instead that disrupting caveolae promotes activation of ICl,swell by reducing the membrane reserves of the cardiac myocyte.If ICl,swell which normally limits swelling during hyposmotic challenge. Because ICl,swell is activated more readily when there are less caveolae, we see a potentiation of the shortening response to swelling which reflects ICl,swell-induced action potential abbreviation  but the negative inotropic effect of swelling  would be less . Furthermore, because MBCD affects both caveolar structure and Cav content , MBCD dot we saw . If non-ICl,swell activation in the cardiac cell, and highlights a general mechanism by which caveolae could modulate mechanosensitive channel activation.This work is the first to show evidence for a role of caveolae in All animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and conforms to the Recommendation from the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. Animals were humanely killed by schedule 1 methods.et al.Myocytes were isolated enzymatically from the hearts of male Wistar rats (250–280 g) according to the method described by Calaghan g. Cell pellets were fixed overnight in 2.5% glutaraldehyde in isotonic or hypotonic solution as appropriate, post-fixed with 1% osmium tetroxide in 100 mM cocadylate buffer and dehydrated through an ethanol series. Cells were embedded in araldite and ultra thin sections (∼100 nm) were stained with 20% uranyl acetate (30 min) and Reynolds lead citrate (30 min) in the presence of 1 M NaOH. Sections were observed using a JEOL 1200 transmission electron microscope.After treatment with isotonic or hypotonic solution for 15 min, a pellet of cells was formed by rapid centrifugation of the cell suspension at 80 Caveolae were defined as flask-shaped invaginations (‘open’ caveolae) or subsarcolemmal vesicles (‘closed’ caveolae) with a size between ≈50 and 100 nm in diameter.2CO3 (pH 11.0) containing 0.5 mM EDTA and 1% protease inhibitor cocktail (Sigma). Samples were homogenised , then sonicated  3 times each for 20 s at full power. Approximately 2 ml of homogenate was mixed with an equal volume of 90% sucrose in MES-buffered saline  to form a 45% sucrose solution. A discontinuous sucrose gradient was created by layering on to this a further 4 ml each of 35% and 5% sucrose solution . Gradients were centrifuged for 17 h at 280,000 g (Beckman SW40Ti rotor) at 4°C. A total of 12 fractions (each 1 ml) were collected following fractionation. Cav 1 and Cav 3 were measured by Western blotting  following SDS-PAGE Preparations were fractionated using detergent-free methods as we have described previously Solutions) at 22–24°C. For measurement of cell volume, cells were quiescent; for assessment of shortening they were field-stimulated at 0.5 Hz and cell length was monitored using an edge-detection system (Crescent Electronics). Cell volume was calculated from a video image of the cell using the relationship V = (ΠwdL)/4  2+(w/6)2)SA = 2ΠL (√((w/2).Isolated myocytes were placed in an experimental chamber on the stage of an inverted microscope and continually perfused with a HEPES-based solution . Hypotonic solution (Hypo) had the same composition and pH as the isotonic standard physiological solution except that 113 mM of sucrose was omitted . Cells were exposed to hypotonic solution for 10–15 min. For lysis experiments, hypotonic solution contained (mM): CaCl2, 1; MgCl2, 1. The pH was adjusted to 7.4 .Isotonic physiological HEPES-based solution (Iso) contained (mM): NaCl, 57; HEPES, 5; NaFor experiments to determine the effect of MBCD on caveolar morphology , control


Tretinoin is considered the benchmark prescription topical therapy for improving fine facial wrinkles, but skin tolerance issues can affect patient compliance. In contrast, cosmetic antiwrinkle products are well tolerated but are generally presumed to be less efficacious than tretinoin.To compare the efficacy of a cosmetic moisturizer regimen vs. a prescription regimen with 0·02% tretinoin for improving the appearance of facial wrinkles.n=99) used a sun protection factor (SPF) 30 moisturizing lotion containing 5% niacinamide, peptides and antioxidants, a moisturizing cream containing niacinamide and peptides, and a targeted wrinkle product containing niacinamide, peptides and 0·3% retinyl propionate. Subjects on the prescription regimen (n=97) used 0·02% tretinoin plus moisturizing SPF 30 sunscreen. Subject cohorts (n=25) continued treatment for an additional 16 weeks. Changes in facial wrinkling were assessed by both expert grading and image analysis of digital images of subjects’ faces and by self-assessment questionnaire. Product tolerance was assessed via clinical erythema and dryness grading, subject self-assessment, and determinations of skin barrier integrity  and stratum corneum protein changes.An 8-week, randomized, parallel-group study was conducted in 196 women with moderate to moderately severe periorbital wrinkles. Following 2 weeks washout, subjects on the cosmetic regimen (The cosmetic regimen significantly improved wrinkle appearance after 8 weeks relative to tretinoin, with comparable benefits after 24 weeks. The cosmetic regimen was significantly better tolerated than tretinoin through 8 weeks by all measures.An appropriately designed cosmetic regimen can improve facial wrinkle appearance comparably with the benchmark prescription treatment, with improved tolerability. Topical tretinoin is considered a benchmark treatment for the mitigation of fine facial wrinkles.5In contrast to the irritancy potential of retinoid therapies, cosmetic and cosmeceutical antiwrinkle products are generally well tolerated by the skin and are pleasant for patients to use. While there are few published reports of direct comparative studies,To determine whether significant wrinkle reduction efficacy comparable with that of a prescription could be achieved using cosmetic products, a treatment regimen was developed containing niacinamide, the palmitoyl peptides palmitoyl-lysine-threonine and palmitoyl-lysine-threonine-threonine-lysine-serine ,ad libidum and a facial moisturizer  twice daily in place of their regular cleansing and moisturizing products. After the preconditioning period, subjects were randomly assigned to either the NPP regimen or the tretinoin regimen for 8 weeks of treatment. A computer-generated randomization list was drawn up by the study statistician and was given to the study site for treatment allocation. Prior to study start, 25 self-selected subjects (cohort) on each product regimen agreed to continue treatment for an additional 16 weeks. Written informed consent for participation in this study was obtained from all subjects in accordance with the Helsinki II declaration, and the protocol was approved by an Institutional Review Board .An 8-week, randomized, parallel-group facial appearance study was performed in Cincinnati, OH, U.S.A. from February to August 2008 in 196 women aged 40–65 years who were neither pregnant nor lactating. Eligible subjects had Fitzpatrick skin types I–III and moderate to moderately severe periorbital wrinkles on both sides of their face. Wrinkle severity was assessed using a six-point ordinal photonumeric scale . SubjectSubjects were instructed to dose both product regimens as per the manufacturer’s package instructions or, when appropriate, investigator guidance. Subjects assigned to the NPP regimen used a daytime sun protection factor (SPF) 30 lotion  in the morning, and a night cream  in the evening, both over the entire face. In addition, these subjects applied a wrinkle treatment  twice daily, that is, prior to the daytime SPF 30 lotion and the night cream, on areas of concern, as determined by the subject. Improvement in the appearance of fine lines and wrinkles was measured by expert visual grading of high-resolution digital images using the Rapid Evaluation of Anti-aging Leads (REAL 3.0) systemIn addition, each subject completed a self-assessment questionnaire containing 14 questions evaluating nine efficacy attributes  using a 10-point continuous scale at baseline and at 2, 4, 6 and 8 weeks in the entire study population and additionally at 16 and 24 weeks in the cohort.Subject tolerability to each regimen was measured by expert clinical grading of erythema and dryness using a six-point scale and by a subject self-assessment questionnaire using a four-point scale at baseline and at 2, 4, 6 and 8 weeks in the entire population and additionally at 16 and 24 weeks in the cohort. Skin barrier integrity was measured by transepidermal water loss (TEWL) at two sites on the face using a Vapometer SWL-2 .D-Squame® tape strips  from the right cheek were collected at baseline and at 8 and 24 weeks, and analysed for the amount of soluble protein, human serum albumin (HSA), involucrin, keratin 6, keratins 1, 10 and 11, fibronectin and cortisol via immunoassay .2 tests. Results were considered significant if P<0·05 (two-sided). Ninety-three subjects would permit detection of a mean wrinkle grade difference of 0·28 at 95% significance (two-sided) with an assumed SD of 0·95 and 80% power. Assuming a 5% drop-out rate, 98 subjects for each group  were required.The primary analysis variable was expert visual grading for the change in appearance of fine lines and wrinkles at 8 weeks. For post-treatment analysis, expert visual grades for the change in appearance of fine lines and wrinkles, change from baseline for wrinkle area fraction, erythema, dryness, self-assessment efficacy questions, and protein analytes from D-Squame® tape strips  were all analysed, separately, via an analysis of covariance model with age and baseline wrinkle grade as covariates using SAS version 9.1 software . Self-assessment tolerability questions were analysed using χOf the 97 tretinoin regimen subjects and 99 NPP regimen subjects who started the study, 93 and 97 subjects, respectively, completed the study. One subject in the tretinoin regimen group withdrew due to redness and dryness; three subjects in the tretinoin regimen group and two in the NPP regimen group were withdrawn due to noncompliance. Five subjects  were excluded from the REAL image analysis due to image quality issues . P=0·05, tretinoin regimen; P<0·01, NPP regimen). The NPP regimen gave significantly greater improvement than the tretinoin regimen . Also, a significantly higher percentage of subjects on the NPP regimen responded substantially to treatment (mean grade ≥ 2) as compared with the tretinoin regimen . For perspective, a +2 grade would indicate visible improvement in at least one to three small lines and/or 15–25% of areas of crepiness or cross-hatching. Examples of subjects showing substantial responses to each treatment regimen are shown in After 8 weeks of treatment, the appearance of facial fine lines and wrinkles improved in both groups as measured by expert visual grading of the REAL images . The mean percentage reduction from baseline in wrinkle area fraction was greater for the NPP regimen group vs. the tretinoin regimen group .After 8 weeks of treatment, the stratum corneum barrier in the tretinoin regimen group was significantly compromised, with TEWL increasing by 5·9 g mbaseline . TEWL vaP=0·01). Erythema in the NPP regimen group was lower at 4, 6 and 8 weeks compared with 2 weeks and was no longer statistically significantly different from baseline. At 6 and 8 weeks, erythema did decrease in the tretinoin regimen but remained significantly higher than at baseline.After 2 weeks of treatment, both product regimen groups exhibited a significant increase in erythema from baseline . After 4P<0·01 for all).At 2, 4, 6 and 8 weeks, the tretinoin regimen group showed significantly more skin dryness from baseline . There wThe results of the subjects’ self-assessments of product tolerability were consistent with the expert erythema and dryness results (data not shown). Compared with the NPP regimen group, after 2 weeks of treatment significantly more subjects in the tretinoin regimen group experienced itchiness, redness/rash and peeling/flaking, which is consistent with known side-effects of tretinoin.There were significant differences between the product regimens for each of the four stratum corneum protein analytes after 8 weeks of treatment . Solublen=25 in each group) who continued on treatment for an additional 16 weeks, 48  completed the entire 24 weeks. As seen in P<0·01 for both). Consistent with the 8-week results for the cohort, after 24 weeks of treatment there were no significant differences between the NPP regimen and the tretinoin regimen by either measure . There was a statistically significant difference in change in TEWL between the two groups at 8 and 24 weeks  but was significantly improved in the NPP regimen group (P<0·01).Of the 50 subjects . After 16 weeks of treatment, erythema in the subset of the tretinoin regimen group was not different from baseline, but at 24 weeks of treatment there was a significant decrease in erythema. Skin dryness in the tretinoin regimen cohort was not significantly different from baseline after 16 and 24 weeks of treatment.After 16 and 24 weeks of treatment, the subset of the NPP regimen group exhibited a nonsignificant decrease from baseline in erythema and dryness . In addition, the NPP regimen subjects did not report any significant irritation after 16 and 24 weeks of treatment; the tretinoin regimen subjects did report significant peeling and flaking at 16 weeks but no significant irritation after 24 weeks (data not shown).P=0·59) but significantly reduced relative to baseline in the NPP regimen group. Involucrin levels remained not significantly different from baseline following 24 weeks use of the NPP regimen; however, levels of involucrin in the tretinoin regimen group decreased between 8 and 24 weeks, which is consistent with the TEWL and irritation data showing accommodation to tretinoin treatment with longer term use. Overall, the NPP regimen group had significantly lower levels of soluble protein, HSA and involucrin and significantly higher levels of keratins 1, 10 and 11 than the tretinoin regimen group at 24 weeks.After 8 weeks, protein analyte changes in the cohort were consistent with those in the full population . After 2To our knowledge, this is the first of its kind long-term clinical study comparing a cosmetic anti-ageing regimen against a recognized prescription topical treatment for improving the appearance of facial wrinkling. The NPP regimen, which contained cosmetic ingredients known to affect the appearance of wrinkles, along with moisturizing and skin barrier building components, comparably improved the appearance of fine lines and wrinkles relative to the prescription tretinoin regimen as measured by both expert grading and image analysis in a long-term study, with better tolerability. Improvements in skin texture with topical niacinamide treatment have been reported and linked to enhanced barrier function,Another observation is that barrier function restoration and stabilization of stratum corneum repair mechanisms may occur at different rates, as TEWL values in the tretinoin cohort had returned to baseline by week 24 but some marker protein levels had not.We recognize that there are limitations in this study. Tretinoin is more irritating to the skin and the products tested in the NPP regimen are more moisturizing. Additionally, while the evaluators were blinded to treatment, the subjects were not which could have affected the subjects' self-assessments. Finally, a larger 24-week cohort group size would have provided more statistical power for more robust conclusions. These limitations notwithstanding, the study data, from both objective and subjective assessments, provided consistent results and showed that the new NPP regimen improved the appearance of fine lines and wrinkles similarly to the tretinoin regimen, with fewer side-effects.The results of this study show that the efficacy of a prescription product for improving the appearance of facial fine lines and wrinkles can be achieved with an appropriately designed cosmetic regimen, while providing additional benefits in aesthetics, skin tolerance and potential patient compliance.