Guilherme B. Bubols

The Antioxidant Activity of Coumarins and Flavonoids

Coumarins and flavonoids are heterocyclic molecules that have been associated with beneficial effects on human health, such as reducing the risk of cancer, diabetes, cardiovascular and brain diseases. These effects are thought to be related to the radical scavenging effect, due to their antioxidant activities, along with other possible mechanisms, such as anti-inflammatory properties and interaction with several enzymes. Over the past two decades, there have been an increasing number of publications on coumarins and flavonoids, which demonstrate the importance of understanding the chemistry behind the antioxidant activities of both natural and synthesized compounds, considering the benefits from their dietary ingestion as well as pharmacological use. This work aims to review the antioxidant effects of coumarin and flavonoid molecules in humans and the structural aspects that contribute to these effects.

Biomarkers of occupational exposure to air pollution, inflammation and oxidative damage in taxi drivers

Exposure to environmental pollutants has been recognised as a risk factor for cardiovascular events. 1-hydroxypyrene (1-OHP) is a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) from traffic-related air pollution. Experimental studies indicate that PAH exposure could be associated with inflammation and atherogenesis. Thus, the purpose of this study was to evaluate whether the biomarker of PAH exposure is associated with biomarkers of inflammation and oxidative stress and if these effects modulate the risk of developing cardiovascular diseases in workers exposed to air pollution. This study included 60 subjects, comprising 39 taxi drivers and 21 non-occupationally exposed persons. Environmental PM2.5 and benzo[a]pyrene (BaP) levels, in addition to biomarkers of exposure and oxidative damage, were determined. Inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α, IFN-γ and hs-CRP) and serum levels of oxidised LDL (ox-LDL), auto-antibodies (ox-LDL-Ab) and homocysteine (Hcy) were also evaluated. PM2.5 and BaP exhibited averages of 12.4±6.9 μg m(-3) and 1.0±0.6 ng m(-3), respectively. Urinary 1-OHP levels were increased in taxi drivers compared to the non-occupationally exposed subjects (p<0.05) and were positively correlated with pro-inflammatory cytokines and negatively correlated with antioxidants. Furthermore, taxi drivers had elevated pro-inflammatory cytokines, biomarkers of oxidative damage, and ox-LDL, ox-LDL-Ab and Hcy levels, although antioxidant enzymes were decreased compared to the non-occupationally exposed subjects (p<0.05). In summary, our findings indicate that taxi drivers showed major exposure to pollutants, such as PAHs, in relation to non-occupationally exposed subjects. This finding was associated with higher inflammatory biomarkers and Hcy, which represent important predictors for cardiovascular events. These data suggest a contribution of PAHs to cardiovascular diseases upon occupational exposure.

Genotoxicity and oxidative stress in gasoline station attendants

We evaluated genotoxic effects of exposure to low levels of benzene, a class I human carcinogen, among gasoline station attendants (GSA). Oxidative stress and the protective effects of antioxidants on DNA damage were also analyzed. Although exposures were below ACGIH (American Conference of Governmental Industrial Hygienists) limits, the GSA group presented higher DNA damage indices and micronucleus frequencies, increased oxidative protein damage, and decreased antioxidant capacity relative to the control group. Duration of benzene exposure was correlated with DNA and protein damage. The biomarkers evaluated in this work may provide early signals of damage in subjects occupationally exposed to benzene.

Evaluation of genotoxicity in workers exposed to benzene and atmospheric pollutants.

Gas station attendants and taxi drivers are occupationally exposed to xenobiotics which may be harmful to their health. Atmospheric pollutants and benzene can lead to DNA damage. Genotoxicity and mutagenicity assays can be used to evaluate the effects of these pollutants. We have evaluated genotoxicity and mutagenicity in workers occupationally exposed to xenobiotics, by application of the 8-hydroxy-2-deoxyguanosine (8-OHdG), comet, and micronucleus (MN) assays. Biomarkers of benzene and carbon monoxyde exposure were also measured: urinary t,t-muconic acid (t,t-MA) and carboxyhaemoglobin (COHb) in whole blood, respectively. The study groups comprised 43 gas station attendants (GSA), 34 taxi drivers (TD), and 22 persons without known occupational exposures (NE). Levels of t,t-MA in the GSA group were significantly elevated compared to the NE group (p<0.001), however these levels were below of levels established by ACGIH (American Conference of Governmental Industrial Hygienists). COHb levels were not significantly different between the TD and NE groups (p>0.05). DNA damage index (DI) and 8-OHdG levels were significantly higher for both the GSA and TD groups, compared to the NE group (p<0.001), but MN frequencies were not elevated. Spearman correlation analysis showed that the frequency of MN was positively correlated with 8-OHdG. A positive correlation between DNA DI levels and 8-OHdG was also observed. In conclusion, our results indicated that low levels of occupational exposure to benzene and atmospheric pollutants may be linked to genotoxicity and oxidative DNA damage.

Cognitive deficits and ALA-D-inhibition in children exposed to multiple metals

Children are especially vulnerable to adverse effects of multiple metals exposure. The aim of this study was to assess some metals concentrations such as lead (Pb), arsenic (As), chromium (Cr), manganese (Mn) and iron (Fe) in whole blood, serum, hair and drinking water samples using inductively coupled plasma-mass spectrometry (ICP-MS) in rural and urban children. In addition, evaluate the adverse effects of multiple metals exposure on cognitive function and δ-aminolevulinate dehydratase (ALA-D) activity. The cognitive ability assessment was performed by the Ravens Colored Progressive Matrices (RCPM) test. The ALA-D activity and ALA-D reactivation index (ALA-RE) activity with DTT and ZnCl2 also were determined. Forty-six rural children and 23 urban children were enrolled in this study. Rural children showed percentile IQ scores in the RCPM test significantly decreased in relation to urban children. According to multiple linear regression analysis, the Mn and Fe in hair may account for the cognitive deficits of children. Manganese and Fe in hair also were positively correlated with Mn and Fe in drinking water, respectively. These results suggest that drinking water is possibly a source of metals exposure in children. ALA-D activity was decreased and ALA-RE with DTT and ZnCl2 was increased in rural children in comparison to urban children. Moreover, ALA-D inhibition was correlated with Cr blood levels and ALA-RE/DDT and ALA-RE/ZnCl2 were correlated with levels of Cr and Hg in blood. Thus, our results indicated some adverse effects of childrens exposure to multiple metals, such as cognitive deficits and ALA-D inhibition, mainly associated to Mn, Fe, Cr and Hg.

Development and validation of a food frequency questionnaire for consumption of polyphenol‐rich foods in pregnant women

Previous studies have shown that maternal consumption of polyphenol-rich foods after the third trimester of pregnancy may interfere with the anatomical and functional activity of the fetal heart as, to our knowledge, there are no validated instruments to quantify total polyphenols in pregnant women. The aim of this study was evaluate the reproducibility and validity of a food frequency questionnaire (FFQ), with 52 items, to assess the intake of polyphenol-rich foods in pregnant women in Brazil. This cross-sectional study included 120 pregnant women who participated in nutritional interviews in two moments. The intake of polyphenols estimated by the developed FFQ was compared with the average of two 24-h recalls (24HR), with the average intake measured by a 3-day food diary (D3days) and with the urinary excretion of total polyphenols. The triangular method was applied to calculate Pearsons correlation coefficients, intraclass correlation and Bland-Altman plots for the FFQ, using an independent biochemical marker, in addition to classification by quarters of consumption. The questionnaires were log transformed, adjusted for body mass index and gestational age. The adjustment for energy was applied only of 24HR and D3days. Analysis of the reproducibility between the FFQ showed a very high correlation (r = 0.72; P < 0.05). A low but significant association was observed between the FFQ and urinary excretion (0.23; P = 0.01). The association between the dietary survey methods was moderate to very high (r = 0.36 to r = 0.72; P < 0.001). In conclusion, this questionnaire showed reproducibility and validity for the quantification of consumption of total polyphenols in pregnant women.

Evaluation of the Antioxidant Capacity of Synthesized Coumarins

Coumarins are secondary metabolites that are widely distributed within the plant kingdom, some of which have been extensively studied for their antioxidant properties. The antioxidant activity of coumarins assayed in the present study was measured by different methods, namely the 1,1-diphenyl-2-picryl-hydrazyl (DPPH•) method, cyclic voltammetry and the antioxidant capacity against peroxyl radicals (ACAP) method. The 7,8-dihydroxy-4-methylcoumarin (LaSOM 78), 5-carboxy-7,8-dihydroxy-4-methylcoumarin (LaSOM 79), and 6,7-dihydroxycoumarin (Esculetin) compounds proved to be the most active, showing the highest capacity to deplete the DPPH radicals, the highest antioxidant capacity against peroxyl radicals, and the lowest values of potential oxidation.

Early hematological and immunological alterations in gasoline station attendants exposed to benzene

INTRODUCTION Elucidation of effective biomarkers may provide tools for the early detection of biological alterations caused by benzene exposure and may contribute to the reduction of occupational diseases. This study aimed to assess early alterations on hematological and immunological systems of workers exposed to benzene. METHODS Sixty gasoline station attendants (GSA group) and 28 control subjects were evaluated. Environmental and biological monitoring of benzene exposure was performed in blood and urine. The potential effect biomarkers evaluated were δ-aminolevulinate dehydratase (ALA-D) activity, CD80 and CD86 expression in lymphocytes and monocytes, and serum interleukin-8 (IL-8). The influence of confounding factors and toluene co-exposure were considered. RESULTS Although exposures were below ACGIH (American Conference of Governmental Industrial Hygienists) limits, reduced ALA-D activity, decreased CD80 and CD86 expression in monocytes and increased IL-8 levels were found in the GSA group compared to the control subjects. Furthermore, according to multiple linear regression analysis, benzene exposure was associated to a decrease in CD80 and CD86 expression in monocytes. CONCLUSIONS These findings suggest, for the first time, a potential effect of benzene exposure on ALA-D activity, CD80 and CD86 expression, IL-8 levels, which could be suggested as potential markers for the early detection of benzene-induced alterations.

Simultaneous quantification of lycopene, β-carotene, retinol and α -tocopherol in plasma after a simple extraction procedure: stability study and application to human volunteers

Um metodo para quantificacao simultânea de licopeno, β-caroteno, retinol e α-tocoferol por cromatografia liquida de alta eficiencia (HPLC) com deteccao no visivel/fluorescente e eluicao isocratica foi otimizado e validado. O metodo consiste de extracao liquido-liquido rapida e simples e posterior quantificacao do sobrenadante extraido por HPLC. Aliquotas de plasma foram estocadas a –20°C por tres meses para estudo da estabilidade. Aplicacao metodologica foi realizada em amostras fornecidas por pintores e individuos nao expostos a tintas (n = 75). O ensaio foi linear para todas as vitaminas analisadas (r > 0,99). Precisoes intradia e interdia apresentaram coeficiente de variacao (CV) menor que 5%. Exatidoes variaram de 0,29 a –5,80% e recuperacoes entre 92,73 e 101,97%. Amostras de plasma e sobrenadante extraido foram estaveis por ate 60 dias a –20°C. Foi demonstrada uma diminuicao significativa nas concentracoes de licopeno, β-caroteno e retinol em individuos expostos quando comparados com os nao-expostos (p 0.99). Intra- and inter-run precisions were obtained with coefficient of variation smaller than 5%. The accuracies ranged from 0.29 to –5.80% and recoveries between 92.73 and 101.97%. Plasma samples and extracted supernatants were stable for 60 days at –20°C. A significant decrease of lycopene, β-carotene and retinol concentrations in plasma from exposed individuals compared to non-exposed individuals (p < 0.05) was observed. The method is simple, reproducible, precise, accurate and sensitive, and can be routinely utilized in clinical laboratories.

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ.