Enzymatic synthesis of bromo- and chlorocarbazoles and elucidation of their structures by molecular modeling.

3-Chlorocarbazole, 3,6-dichlorocarbazole, dibromocarbazole, and 1,3,6,8-tetrabromocarbazole are emerging environmental contaminants which have been detected recently in water, sediment, and soil samples. However, their sources and occurrence have not been explained. Here, we report an enzymatic synthesis of bromo- and chlorocarbazoles by chloroperoxidase from Caldariomyces fumago in water. Density functional theory (DFT) method was used to predict the most stable products. Carbazole and chloroperoxidase were assayed in vitro in the presence of hydrogen peroxide, bromide, and chloride ions in different substrate ratio treatments against constant and varying enzyme concentrations. Halogenated carbazoles formed were identified by high-resolution gas chromatography coupled to mass spectrometry. In all treatments, bromination and chlorination took place, but the composition and concentration of compounds formed varied from one treatment to another. Mono-, di-, tri-, and tetra-substituted bromo- and chlorocarbazoles which include the reported environmental contaminants were synthesized. 3-Substituted and 3,6-substituted congeners were relatively higher in concentration. Enzyme concentration did not favor preferential formation of any of the compounds synthesized. However, their synthesis was influenced by halide concentration. Congeners with bromine and chlorine at position of C-3, C-3,6, C-1,3,6, and C-1,3,6,8 were calculated as the stable intermediate sigma complexes by DFT method. Regioselectivity in halogenation is discussed and hypothesis of the likely stable products in the environment explained. This study provides evidence that bromo- and chlorocarbazoles reported previously can be formed enzymatically in the environment, demonstrating the need to consider aromatic pollutants transformation and their potential toxicity enhancements in the management of water pollution and contaminated sites

Photodegradative fate and potential phototoxic products of bromocarbazoles and chlorocarbazoles in water.

Bromocarbazoles and chlorocarbazoles are emerging environmental contaminants that have been reported to be persistent and possessing dioxin-like toxicity; however, their photodegradative fate in water is unknown. The photodegradation of 3-bromocarbazole, 3-chlorocarbazole, and 3,6-dichlorocarbazole was determined in ultrapure water. They proceeded by direct photolysis and followed first-order kinetics. The rate constants (k) were 0.4838, 0.3454, and 0.4422 h(-1) corresponding to half-lives (t 1/2) 1.81, 2.01, and 1.62, while the quantum yields (Ф) were 0.232, 0.180, and 0.295 respectively. The maximum wavelengths of absorption (λ max) were in the near ultraviolet region (295, 296, 299, and 301 nm) implying these compounds are likely to degrade slowly under sunlight in natural aquatic environment. The molar extinction coefficients (ε) determined in acetonitrile were 18,573, 17,028, 13,385, and 14,010 L mol(-1) cm(-1), respectively, the latter being 3,6-dibromocarbazole. A bathochromic shift was observed with halogen addition on their respective mono-substituted congeners. Bromocarbazoles were observed to degrade faster in water than chlorocarbazoles. In addition, photodegradation was estimated to proceed faster in summer than in winter, in natural water system at 50° N latitude. In the absence of light, hydrolytic degradation occurred but proceeded very slowly. Hexahydroxybenzene and trihydroxycarbazole were positively identified as the likely photoproducts with the former being a known toxic compound. Dehalogenation, oxidative cleavage, hydroxylation, and hydrolysis are suggested as the major photodegradation mechanisms in water, yielding phototoxic products that may be of enhanced toxicity than the parent compounds

The fingerprints of dioxin-like bromocarbazoles and chlorocarbazoles in selected forest soils in Germany.

The occurrence of bromocarbazoles and chlorocarbazoles was studied in 86 forest soil samples from different regions in Germany. Carbazole, 3-chlorocarbazole, 3-bromocarbazole and 3,6-dibromocarbazole were qualitatively detected in the humic layer of 59 soil samples with bromocarbazoles reported here for the first time in soil. Furthermore, the halogenated carbazoles, PCDD/Fs and PCBs were detected in the humic and mineral soil horizons (0-5 cm and 5-10 cm) of a subset of 11 soil samples subjected to quantitative analysis. Concentrations ranged from 0.6 to 267.6 ng/g (carbazole); 0.2-7.2 ng/g (3-bromocarbazole); 0.0-9.1 ng/g (3-chlorocarbazole); 0.2-19.8 ng/g (3,6-dibromocarbazole); 0.4-67.6 ng/g (3,6-dichlorocarbazole); 0.0-0.7 ng/g (PCDDs); 0.0-0.3 ng/g (PCDFs) and 0.0-33.7 ng/g (PCBs). Concentrations decreased with depth and correlated positively to total organic carbon (TOC). When it was based on TOC%, an increase in concentration with depth was observed in most soil samples. With respect to dioxin-like toxicity, 3-bromocarbazole, 3-chlorocarbazole, 3,6-dibromocarbazole and 3,6-dichlorocarbazoles caused induction of CYP1A1-dependent EROD activity in HII4E rat hepatoma cell line. Their relative effect potency after 72 h exposure ranged from 0.00005 to 0.00013 and was directly related to the degree of halogenation with 2,3,7,8-tetrachlorodibenzo-p-dioxin as reference. Furthermore, their contribution to overall soil dioxin-like toxicity was not significant in comparison to PCDD/Fs and PCBs though the sum toxic equivalency was limited to three halogenated carbazole congeners. Bromocarbazoles and chlorocarbazoles are emerging dioxin-like toxic environmental contaminants with potential for wide distribution occurring simultaneously with PCDD/Fs and PCBs

Persistence and dioxin-like toxicity of carbazole and chlorocarbazoles in soil.

Halogenated carbazoles have recently been detected in soil and water samples, but their environmental effects and fate are unknown. Eighty-four soil samples obtained from a site with no recorded history of pollution were used to assess the persistence and dioxin-like toxicity of carbazole and chlorocarbazoles in soil under controlled conditions for 15 months. Soil samples were divided into two temperature conditions, 15 and 20 °C, both under fluctuating soil moisture conditions comprising 19 and 44 drying-rewetting cycles, respectively. This was characterized by natural water loss by evaporation and rewetting to -15 kPa. Accelerated solvent extraction (ASE) and cleanup were performed after incubation. Identification and quantification were done using high-resolution gas chromatogram/mass spectrometer (HRGC/MS), while dioxin-like toxicity was determined by ethoxyresorufin-O-deethylase (EROD) induction in H4IIA rat hepatoma cells assay and multidimensional quantitative structure-activity relationships (mQSAR) modelling. Carbazole, 3-chlorocarbazole and 3,6-dichlorocarbazole were detected including trichlorocarbazole not previously reported in soils. Carbazole and 3-chlorocarbazole showed significant dissipation at 15 °C but not at 20 °C incubating conditions indicating that low temperature could be suitable for dissipation of carbazole and chlorocarbazoles. 3,6-Dichlorocarbazole was resistant at both conditions. Trichlorocarbazole however exhibited a tendency to increase in concentration with time. 3-Chlorocarbazole, 3,6-dibromocarbazole and selected soil extracts exhibited EROD activity. Dioxin-like toxicity did not decrease significantly with time, whereas the sum chlorocarbazole toxic equivalence concentrations (∑TEQ) did not contribute significantly to the soil assay dioxin-like toxicity equivalent concentrations (TCDD-EQ). Carbazole and chlorocarbazoles are persistent with the latter also toxic in natural conditions

Population effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and non-typeable Haemophilus influenzae in Kilifi, Kenya: findings from cross-sectional carriage studies.

BACKGROUND: The effect of 7-valent pneumococcal conjugate vaccine (PCV) in developed countries was enhanced by indirect protection of unvaccinated individuals, mediated by reduced nasopharyngeal carriage of vaccine-serotype pneumococci. The potential indirect protection of 10-valent PCV (PCV10) in a developing country setting is unknown. We sought to estimate the effectiveness of introduction of PCV10 in Kenya against carriage of vaccine serotypes and its effect on other bacteria. METHODS: PCV10 was introduced into the infant vaccination programme in Kenya in January, 2011, accompanied by a catch-up campaign in Kilifi County for children aged younger than 5 years. We did annual cross-sectional carriage studies among an age-stratified, random population sample in the 2 years before and 2 years after PCV10 introduction. A nasopharyngeal rayon swab specimen was collected from each participant and was processed in accordance with WHO recommendations. Prevalence ratios of carriage before and after introduction of PCV10 were calculated by log-binomial regression. FINDINGS: About 500 individuals were enrolled each year (total n=2031). Among children younger than 5 years, the baseline (2009-10) carriage prevalence was 34% for vaccine-serotype Streptococcus pneumoniae, 41% for non-vaccine-serotype Streptococcus pneumoniae, and 54% for non-typeable Haemophilus influenzae. After PCV10 introduction (2011-12), these percentages were 13%, 57%, and 40%, respectively. Adjusted prevalence ratios were 0·36 (95% CI 0·26-0·51), 1·37 (1·13-1·65), and 0·62 (0·52-0·75), respectively. Among individuals aged 5 years or older, the adjusted prevalence ratios for vaccine-serotype and non-vaccine-serotype S pneumoniae carriage were 0·34 (95% CI 0·18-0·62) and 1·13 (0·92-1·38), respectively. There was no change in prevalence ratio for Staphylococcus aureus (adjusted prevalence ratio for those <5 years old 1·02, 95% CI 0·52-1·99, and for those ≥5 years old 0·90, 0·60-1·35). INTERPRETATION: After programmatic use of PCV10 in Kilifi, carriage of vaccine serotypes was reduced by two-thirds both in children younger than 5 years and in older individuals. These findings suggest that PCV10 introduction in Africa will have substantial indirect effects on invasive pneumococcal disease. FUNDING: GAVI Alliance and Wellcome Trust

Comparative study of dioxin contamination from forest soil samples (BZE II) by mass spectrometry and EROD bioassay.

Dioxins and dioxin-like compounds can be analyzed by bioanalytical screening methods to evaluate their biotoxicity. In vitro bioassays, based on 7-ethoxyresorufin-O-deethylase (EROD) and the activity of cytochrome P450 1A1 and the aryl hydrogen receptor (AhR) pathway, are employed for the evaluation of bioanalytical equivalents (BEQ) of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) from a wide variety of sample matrices. Here, we present the evaluation of 11 humic soil samples derived from forest stands across Germany and a comparison of the BEQ values against toxic equivalents (TEQ, PCDD/Fs+PCBs) derived by chemical analysis. BEQ values ranged from 8.8 to 34.1 while TEQ values from 13.9 to 60.5 pg/g dry weight. Additional two subsequent mineral layers were analyzed to identify the BEQ/TEQ gradient vertically, showing a TEQ decrease of 85.1 and 93.8 % from the humic to the first and second mineral layers, respectively. For BEQ values, a decrease as well as an increase was detected. BEQ measurements were performed with and without sample clean-up. Omitting clean-up revealed about 20 times increased BEQ values presumably due to non-persistent bioactive compounds not detected by chemical analysis. The results we present suggest that the EROD assay can be used for the screening of large sample quantities for the identification of samples showing dioxin and dioxin-like contaminations even at low levels, which can then be further analyzed by chemical analysis to identify the congener composition. The study also shows that EROD results give a qualitative image of the contamination. EROD seems to be interfered with cross-contaminants specifically for soils with high biological activity as forest layers

Population effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and non-typeable Haemophilus influenzae in Kilifi, Kenya: findings from cross-sectional carriage studies

Background: The effect of 7-valent pneumococcal conjugate vaccine (PCV) in developed countries was enhanced by indirect protection of unvaccinated individuals, mediated by reduced nasopharyngeal carriage of vaccine-serotype pneumococci. The potential indirect protection of 10-valent PCV (PCV10) in a developing country setting is unknown. We sought to estimate the effectiveness of introduction of PCV10 in Kenya against carriage of vaccine serotypes and its effect on other bacteria. Methods: PCV10 was introduced into the infant vaccination programme in Kenya in January, 2011, accompanied by a catch-up campaign in Kilifi County for children aged younger than 5 years. We did annual cross-sectional carriage studies among an age-stratified, random population sample in the 2 years before and 2 years after PCV10 introduction. A nasopharyngeal rayon swab specimen was collected from each participant and was processed in accordance with WHO recommendations. Prevalence ratios of carriage before and after introduction of PCV10 were calculated by log-binomial regression. Findings: About 500 individuals were enrolled each year (total n=2031). Among children younger than 5 years, the baseline (2009–10) carriage prevalence was 34% for vaccine-serotype Streptococcus pneumoniae, 41% for non-vaccine-serotype Streptococcus pneumoniae, and 54% for non-typeable Haemophilus influenzae. After PCV10 introduction (2011–12), these percentages were 13%, 57%, and 40%, respectively. Adjusted prevalence ratios were 0·36 (95% CI 0·26–0·51), 1·37 (1·13–1·65), and 0·62 (0·52–0·75), respectively. Among individuals aged 5 years or older, the adjusted prevalence ratios for vaccine-serotype and non-vaccine-serotype S pneumoniae carriage were 0·34 (95% CI 0·18–0·62) and 1·13 (0·92–1·38), respectively. There was no change in prevalence ratio for Staphylococcus aureus (adjusted prevalence ratio for those <5 years old 1·02, 95% CI 0·52–1·99, and for those ≥5 years old 0·90, 0·60–1·35). Interpretation: After programmatic use of PCV10 in Kilifi, carriage of vaccine serotypes was reduced by two-thirds both in children younger than 5 years and in older individuals. These findings suggest that PCV10 introduction in Africa will have substantial indirect effects on invasive pneumococcal disease. Funding: GAVI Alliance and Wellcome Trust

Effect of ten-valent pneumococcal conjugate vaccine on invasive pneumococcal disease and nasopharyngeal carriage in Kenya: a longitudinal surveillance study

BACKGROUND: Ten-valent pneumococcal conjugate vaccine (PCV10), delivered at 6, 10, and 14 weeks of age was introduced in Kenya in January, 2011, accompanied by a catch-up campaign in Kilifi County for children aged younger than 5 years. Coverage with at least two PCV10 doses in children aged 2-11 months was 80% in 2011 and 84% in 2016; coverage with at least one dose in children aged 12-59 months was 66% in 2011 and 87% in 2016. We aimed to assess PCV10 effect against nasopharyngeal carriage and invasive pneumococcal disease (IPD) in children and adults in Kilifi County. METHODS: This study was done at the KEMRI-Wellcome Trust Research Programme among residents of the Kilifi Health and Demographic Surveillance System, a rural community on the Kenyan coast covering an area of 891 km2. We linked clinical and microbiological surveillance for IPD among admissions of all ages at Kilifi County Hospital, Kenya, which serves the community, to the Kilifi Health and Demographic Surveillance System from 1999 to 2016. We calculated the incidence rate ratio (IRR) comparing the prevaccine (Jan 1, 1999-Dec 31, 2010) and postvaccine (Jan 1, 2012-Dec 31, 2016) eras, adjusted for confounding, and reported percentage reduction in IPD as 1 minus IRR. Annual cross-sectional surveys of nasopharyngeal carriage were done from 2009 to 2016. FINDINGS: Surveillance identified 667 cases of IPD in 3 211 403 person-years of observation. Yearly IPD incidence in children younger than 5 years reduced sharply in 2011 following vaccine introduction and remained low (PCV10-type IPD: 60·8 cases per 100 000 in the prevaccine era vs 3·2 per 100 000 in the postvaccine era [adjusted IRR 0·08, 95% CI 0·03-0·22]; IPD caused by any serotype: 81·6 per 100 000 vs 15·3 per 100 000 [0·32, 0·17-0·60]). PCV10-type IPD also declined in the post-vaccination era in unvaccinated age groups (<2 months [no cases in the postvaccine era], 5-14 years [adjusted IRR 0·26, 95% CI 0·11-0·59], and ≥15 years [0·19, 0·07-0·51]). Incidence of non-PCV10-type IPD did not differ between eras. In children younger than 5 years, PCV10-type carriage declined between eras (age-standardised adjusted prevalence ratio 0·26, 95% CI 0·19-0·35) and non-PCV10-type carriage increased (1·71, 1·47-1·99). INTERPRETATION: Introduction of PCV10 in Kenya, accompanied by a catch-up campaign, resulted in a substantial reduction in PCV10-type IPD in children and adults without significant replacement disease. Although the catch-up campaign is likely to have brought forward the benefits by several years, the study suggests that routine infant PCV10 immunisation programmes will provide substantial direct and indirect protection in low-income settings in tropical Africa. FUNDING: Gavi, The Vaccine Alliance and The Wellcome Trust of Great Britain