Mixture effects of food-relevant polycyclic aromatic hydrocarbons on the activation of nuclear receptors and gene expression, benzo[a]pyrene metabolite profile and DNA damage in HepaRG cells.

Carcinogenic benzo[a]pyrene (BP) and other non-carcinogenic polycyclic aromatic hydrocarbons (PAH) like fluoranthene (FA) and pyrene (PYR) occur as food contaminants. Molecular effects of BP, FA and PYR in human liver cells were investigated using mixtures occurring in grilled meat. Activation of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) was investigated along with target gene expression. Mixture effects on BP metabolite profile and DNA-damaging potential were studied as biological downstream effects. Compared to BP, FA and PYR activated the AHR only weakly. Mixtures were less efficient than BP. Analysis of CYP1A1 expression showed synergistic induction after co-exposure in HepaRG cells. FA and PYR were strong CAR agonists, whereas BP was less potent. Mixtures containing BP caused a strong decrease of CAR transactivation in line with lower CYP2B6 expression. The BP metabolite profile and BP-induced DNA damage were only weakly affected. PAH mixtures modulate AHR, CAR activation and their target genes. However, these mixture effects appear not to be reflected at the level of downstream events like BP metabolite formation or BP-induced DNA damage. Our study clearly shows that endpoints at all biological levels should be considered for mixture evaluation, instead of drawing conclusions exclusively based on early molecular events.

Ultra-high sensitive analysis of 3-hydroxybenzo[a]pyrene in human urine using GC-APLI-MS.

Urinary 3-hydroxybenzo[a]pyrene (3-OH-BaP) is a known biomarker for human exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH). In this work, a new method for the ultra-sensitive quantification of this biomarker has been developed using the hyphenation of gas chromatography and atmospheric pressure laser ionization-mass spectrometry (GC-APLI-MS). In combination with an advanced sample preparation, a limit of detection (LOD) of 0.6 pg/L was achieved which is an improvement by a factor of at least 28 compared with existing methods. The limit of quantification (LOQ) is 1.8 pg/L. With this set-up 3-OH-BaP could be analyzed in urine samples of 7 smokers and 7 non-smokers. Concentrations ranged from 37 to 270 pg/L for non-smokers and from 374 to 1171 pg/L for smokers. For the first time, 3-OH-BaP was quantifiable in all non-smoker samples as no value was below the LOQ. Correlation of the urinary 3-OH-BaP values with the number of daily smoked cigarettes and with urinary cotinine values shows a clear relationship between 3-OH-BaP content and smoking habits. This innovative analytical method enables monitoring of low levels of the biomarker 3-OH-BaP in urine of non-occupationally exposed individuals including smokers, the general population with background PAH exposure and cohorts of low exposition such as newborns and children.

GC-APLI-MS as a powerful tool for the analysis of BaP-tetraol in human urine.

For the first time gas chromatography (GC) coupled to atmospheric pressure laser ionization-mass spectrometry (APLI-MS) has been applied to the analysis of trans-anti-benzo[a]pyrene-tetraol (BaP-tetraol) formed from anti-benzo[a]pyrene diolepoxide (BPDE), the ultimate carcinogen of benzo[a]pyrene. This tetraol is considered to be an ideal urinary biomarker for polycyclic aromatic hydrocarbon (PAH) exposure as it reflects internal body burden and potentially adverse health effects. Optimization of the derivatization and the instrumental set-up led to an instrumental LOD of 0.5 fg, an improvement of the lowest instrumental LOD reported in literature of 6.4 fg by a factor of 10. The optimized procedure includes derivatization of hydroxyl groups using methyl iodide and cool on-column injection to prevent degradation of the analyte. First measurements of urine samples demonstrate that the method is capable of detecting BaP-tetraol in human urine collected from both smokers and non-smokers. Although results of analysis indicate a certain underestimation compared with literature data, this method can be expected to serve as an excellent method for the analysis of the biomarker BaP-tetraol in the future if an adequate internal standard such as 13C-labeled BaP-tetraol is applied.

Urinary toxicokinetics of di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans following single oral administration.

Phthalates such as di-2-ethylhexyl phthalate (DEHP) were restricted due to their toxic mainly reprotoxic effects. Therefore compounds such as di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) substitute these phthalates and the exposure of humanes to substitutes may occur. Here, kinetic data are presented to assess the exposure of humans. Male and female volunteers excreted nearly the complete orally administered dose (1mg/kg b.w. corresponding to the tolerable daily intake of EFSA) of di-(isononyl)-cyclohexane-1,2-dicarboxylate within 70 h. More than 75% were excreted within 24h. Besides the main metabolite cyclohexane-1,2-dicarboxylic acid (CHDA) quantitated after hydrolysis four further metabolites of DINCH(®) are determined. Cyclohexane-1,2-dicarboxylic acid-mono-(7-hydroxy-4-methyl)octyl ester (OH-MINCH) is the main secondary metabolite with about 14% of the administered dose. Differences in excretion of all metabolites between male and females are small. Based on the generated toxicokinetic data exposure of 20 humans is recalculated from their spot urine sample collected in 2014 and the exposure are clearly below the current tolerable daily intake of 1mg/kg b.w.

Multidrug resistance-associated proteins are involved in the transport of the glutathione conjugates of the ultimate carcinogen of benzo[a]pyrene in human Caco-2 cells.

A wide variety of contaminants are ingested through food, among them the pro-carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) that is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies have revealed that BP phenols are excreted as Phase II metabolites including glucuronides and sulfates. This export is mediated by the breast cancer resistance protein (ABCG2). The ultimate carcinogenic Phase I BP metabolite anti-BP-7,8-dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and excretion of stereoisomeric glutathione conjugates in the presence of an inhibitor of the glutathione-cleaving enzyme γ-glutamyl transpeptidase at the cell surface. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of polarized Caco-2 monolayers. Inhibition studies revealed that the multidrug resistance-associated proteins (ABCCs) are involved in the transport of BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated, thus making it possible to demonstrate that ABCC1 mediates the basolateral and ABCC2 the apical excretion of BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. This finding is equivalent to a transport into feces as well as blood system in the in vivo situation.

Molecular epidemiology of invasive neonatal Streptococcus agalactiae isolates in Germany.

BACKGROUND: Streptococcus agalactiae [group B streptococcus (GBS)] is a well-known cause of invasive infections leading to sepsis and meningitis in neonates. A comprehensive nationwide active surveillance study over 2 years was performed in Germany to describe the molecular epidemiology among 296 invasive neonatal GBS isolates. METHODS: Isolates were typed by pulsed-field gel electrophoresis (PFGE). Typing results were compared with serotypes as well as to clinical data on disease onset, meningitic involvement, and outcome. RESULTS: A remarkable clustering was found with about 60% of all typeable invasive isolates being annotated to one of 7 major PFGE groups, and clusters being nationally widely spread over the whole time period. Despite heterogenic elements, certain PFGE groups were closely related to singular serotypes, especially serotypes V (82%), Ia (84%), and Ib (77%). PFGE groups and serotypes were also partly related to clinical presentation as either early onset disease or late onset disease, and either meningitis or nonmeningitic GBS disease, but not to outcome. CONCLUSIONS: There is a remarkable clonality among invasive GBS isolates that are widely spread geographically and in time; however, no specific clonal lines could be correlated to disease severity and outcome.

Enduring stigma: the long-term effects of incarceration on health.

Although incarceration rates have risen sharply since the 1970s, medical sociology has largely neglected the health effects of imprisonment. Incarceration might have powerful effects on health, especially if it instills stigma, and it could provide sociologists with another mechanism for understanding health disparities. This study identifies some of incarceration's direct and indirect effects and rigorously tests them using the National Longitudinal Survey of Youth. It finds that incarceration has powerful effects on health, but only after release. A history of incarceration strongly increases the likelihood of severe health limitations. Furthermore, any contact with prison is generally more important than the amount of contact, a finding consistent with a stigma-based interpretation. Although this relationship is partly attributable to diminished wage growth and marital instability, the bulk of the effect remains even under the most stringent of specifications, including controls for intelligence and the use of fixed effects, suggesting a far-reaching process with a proliferation of risk factors. The study also finds that incarceration contributes only modestly to racial disparities, that there are few synergistic interactions between incarceration and other features of inequality, including schooling, and that the evidence for a causal effect is much weaker among persistent recidivists and those serving exceptionally long sentences. These study findings are inconsistent with recent speculation; nevertheless, incarceration is an important addition to sociology's research agenda. Exploring incarceration could lead to, among other things, a fruitful synergy among studies on fundamental causes, stigma, and stress.

Concordance between the deduced acetylation status generated by high-speed: real-time PCR based NAT2 genotyping of seven single nucleotide polymorphisms and human NAT2 phenotypes determined by a caffeine assay.

BACKGROUND: The utility of typing single nucleotide polymorphisms (SNPs) for the determination of the N-acetyltransferase 2 (NAT2) acetylation status is a matter of debate. AIMS OF THE STUDY: Evaluation of the concordance between deduced genotype results of seven human NAT2 SNPs generated by Real-time PCR analysis and human NAT2 phenotypes. METHODS: NAT2 phenotypes of 38 Caucasian workers were determined using a suitable caffeine test method. Genomic DNA aliquots were used for the determination of seven human NAT2-specific SNPs (G191A, C282T, T341C, C481T, G590A, A803G, G857A). RESULTS AND CONCLUSIONS: Phenotypic results based on the molar ratio of 5-acetylamino-6-formylamino-3-methyluracil (AFMU)/(AFMU+1-methlyuric acid (1U)+1-methylxanthine (1X)) calculated from excreted caffeine metabolite levels in urine samples with 0.3 as a cut-off point between slow (<0.3) and rapid acetylators (>or=0.3). Twenty-seven samples belonged to the slow (mean 0.13; range: 0.03-0.25), 11 to the rapid (mean: 0.41; range: 0.34-0.48) acetylators. LightCycler analyses revealed 11 different NAT2 variant combinations, whereby *5B/*5B and *5B/*6A or *5A/*6C (each 21%), were the most frequent. The deduced acetylation status of the seven NAT2 SNPs matched perfectly with the 38 results determined by phenotyping. This study showed a 100% concordance between NAT2 phenotypes and the deduced NAT2 genotypes and the suitability of the high-speed NAT2-specific LightCycler analysis in a Caucasian population.

Staphylococcus epidermidis adherence to human amniotic membrane and to human, rabbit, and cat conjunctiva.

PURPOSE: To evaluate Staphylococcus epidermidis adherence to human amniotic membrane (HAM) and compare it with S epidermidis adherence to human, rabbit, and cat conjunctiva in vitro. SETTING: Research laboratory, Loyola University Medical Center, Maywood, Illinois, USA. METHODS: Commercially available HAM (N = 3) was used. Conjunctival specimens from humans, rabbits, and cats (n = 3 each) were processed similarly to HAM. The tissues were exposed to S epidermidis (3 x 10(8) colony-forming units per milliliter) for 0, 5, 30, and 90 minutes, rinsed in sterile saline, and processed for light, scanning (SEM), and transmission (TEM) electron microscopy. Scanning electron microscopy (x2000) was used to quantify adherent bacteria/mm(2) of tissue (SEM photographs = 144). RESULTS: The following mean levels (+/- SD) of adherent S epidermidis/mm(2) were found at 0, 5, 30, and 90 minutes: HAM, 3833 +/- 1570, 9060 +/- 2512, 15,431 +/- 10,752, and 30,315 +/- 14,803, respectively; human conjunctiva, 1493 +/- 672, 7218 +/- 3179, 17,273 +/- 7168, and 19,861 +/- 9624, respectively; rabbit conjunctiva, 3385 +/- 5074, 14,386 +/- 14,569, 15,283 +/- 13,679, and 20,113 +/- 24,016, respectively; and cat conjunctiva, 4032 +/- 2240, 12,345 +/- 3413, 8512 +/- 4032, and 19,214 +/- 5584, respectively. No statistically significant differences were found at any time point (P>.16). CONCLUSION: There was no statistically significant difference in the adherence of S epidermidis to HAM and to human, rabbit, and cat conjunctiva. Bacterial adherence to HAM may be clinically significant.