Adverse childhood experiences and substance misuse in young people in India: results from the multisite cVEDA cohort.

BACKGROUND: Adverse childhood experiences (ACEs) increases vulnerability to externalising disorders such as substance misuse. The study aims to determine the prevalence of ACEs and its association with substance misuse. METHODS: Data from the Consortium on Vulnerability to Externalising Disorders and Addictions (cVEDA) in India was used (n = 9010). ACEs were evaluated using the World Health Organisation (WHO) Adverse Childhood Experiences International Questionnaire whilst substance misuse was assessed using the WHO Alcohol, Smoking and Substance Involvement Screening Test. A random-effects, two-stage individual patient data meta-analysis explained the associations between ACEs and substance misuse with adjustments for confounders such as sex and family structure. RESULTS: 1 in 2 participants reported child maltreatment ACEs and family level ACEs. Except for sexual abuse, males report more of every individual childhood adversity and are more likely to report misusing substances compared with females (87.3% vs. 12.7%). In adolescents, family level ACEs (adj OR 4.2, 95% CI 1.5-11.7) and collective level ACEs (adj OR 6.6, 95% CI 1.4-31.1) show associations with substance misuse whilst in young adults, child level ACEs such as maltreatment show similar strong associations (adj OR 2.0, 95% CI 1.1-3.5). CONCLUSION: ACEs such as abuse and domestic violence are strongly associated with substance misuse, most commonly tobacco, in adolescent and young adult males in India. The results suggest enhancing current ACE resilience programmes and 'trauma-informed' approaches to tackling longer-term impact of ACEs in India. FUNDING: Newton Bhabha Grant jointly funded by the Medical Research Council, UK (MR/N000390/1) and the Indian Council of Medical Research (ICMR/MRC-UK/3/M/2015-NCD-I).

Uncovering selection bias in case-control studies using Bayesian post-stratification.

Case-control studies are particularly prone to selection bias, which can affect odds ratio estimation. Approaches to discovering and adjusting for selection bias have been proposed in the literature using graphical and heuristic tools as well as more complex statistical methods. The approach we propose is based on a survey-weighting method termed Bayesian post-stratification and follows from the conditional independences that characterise selection bias. We use our approach to perform a selection bias sensitivity analysis by using ancillary data sources that describe the target case-control population to re-weight the odds ratio estimates obtained from the study. The method is applied to two case-control studies, the first investigating the association between exposure to electromagnetic fields and acute lymphoblastic leukaemia in children and the second investigating the association between maternal occupational exposure to hairspray and a congenital anomaly in male babies called hypospadias. In both case-control studies, our method showed that the odds ratios were only moderately sensitive to selection bias.

The emerging burden of liver disease in cystic fibrosis patients: A UK nationwide study.

OBJECTIVE: Cystic fibrosis associated liver disease (CFLD) is the third largest cause of mortality in CF. Our aim was to define the burden of CFLD in the UK using national registry data and identify risk factors for progressive disease. METHODS: A longitudinal population-based cohort study was conducted. Cases were defined as all patients with CFLD identified from the UK CF Registry, 2008-2013 (n = 3417). Denominator data were derived from the entire UK CF Registry. The burden of CFLD was characterised. Regression analysis was undertaken to identify risk factors for cirrhosis and progression. RESULTS: Prevalence of CFLD increased from 203.4 to 228.3 per 1000 patients during 2008-2013. Mortality in CF patients with CFLD was more than double those without; cirrhotic patients had higher all-cause mortality (HR 1.54, 95% CI 1.09 to 2.18, p = 0.015). Median recorded age of cirrhosis diagnosis was 19 (range 5-53) years. Male sex, Pseudomonas airway infection and CF related diabetes were independent risk factors for cirrhosis. Ursodeoxycholic acid use was associated with prolonged survival in patients without cirrhosis. CONCLUSIONS: This study highlights an important changing disease burden of CFLD. The prevalence is slowly increasing and, importantly, the disease is not just being diagnosed in childhood. Although the role of ursodeoxycholic acid remains controversial, this study identified a positive association with survival.

Bayesian spatial modelling for quasi-experimental designs: An interrupted time series study of the opening of Municipal Waste Incinerators in relation to infant mortality and sex ratio.

BACKGROUND: There is limited evidence on potential health risks from Municipal Waste Incinerators (MWIs), and previous studies on birth outcomes show inconsistent results. Here, we evaluate whether the opening of MWIs is associated with infant mortality and sex ratio in the surrounding areas, extending the Interrupted Time Series (ITS) methodological approach to account for spatial dependencies at the small area level. METHODS: We specified a Bayesian hierarchical model to investigate the annual risks of infant mortality and sex-ratio (female relative to male) within 10 km of eight MWIs in England and Wales, during the period 1996-2012. We included comparative areas matched one-to-one of similar size and area characteristics. RESULTS: During the study period, infant mortality rates decreased overall by 2.5% per year in England. The opening of an incinerator in the MWI area was associated with -8 deaths per 100,000 infants (95% CI -62, 40) and with a difference in sex ratio of -0.004 (95% CI -0.02, 0.01), comparing the period after opening with that before, corrected for before-after trends in the comparator areas. CONCLUSION: Our method is suitable for the analysis of quasi-experimental time series studies in the presence of spatial structure and when there are global time trends in the outcome variable. Based on our approach, we do not find evidence of an association of MWI opening with changes in risks of infant mortality or sex ratio in comparison with control areas.

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

Delineation of an enhancerlike positive regulatory element in the interleukin-2 receptor alpha-chain gene.

We have delineated a positive regulatory element in the interleukin-2 receptor alpha-chain gene (IL-2R alpha) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-kappa B; however, unlike the immunoglobulin kappa gene kappa B enhancer element, the IL-2R alpha kappa B-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2R alpha kappa B-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2R alpha upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2R alpha promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2R alpha gene transcription.

In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

Uptake of chlorination disinfection by-products; a review and a discussion of its implications for exposure assessment in epidemiological studies.

We have reviewed the relevant issues in the exposure assessment of disinfection by-products (DBPs) of chlorination for epidemiological and health risk assessment. Various DBPs can be detected in drinking water and swimming pools, and the reported levels show a considerable range, but were generally below the current health standard for total trihalomethanes (TTHMs) (100 microg/l). Relatively little information is available on the correlation between the various DBPs in drinking water and in swimming pools. Chloroform was generally, but not always, the most predominant DBP. In epidemiological studies, TTHM levels have been used as an indicator for total DBP load, even though TTHM levels do not always correlate well with individual DPBs. Factors such as residence time, temperature, pH, organic content, including humic and fulvic acid and bromide levels affect the composition and levels of DBPs. Although there are biomarkers of DBPs, mainly for chloroform and more recently for the other volatile trihalomethanes (THMs) and the nonvolatile haloacetic acids (HAAs) such as trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA), they have not been used in epidemiological studies. The THMs have been measured in exhaled breath and serum, while the HAAs have been measured in urine. These biomarkers have been useful to estimate the actual uptake of the DBPs and the relative contribution of various exposure routes. Physiologically based pharmacokinetic (PBPK) models exist for, e.g. chloroform, but their main target organs are the kidney and liver and they have not been used in epidemiological studies. Tap water ingestion, showering, bathing, swimming, boiling water and dishwashing are all activities that have been associated with the uptake of DBPs, and considerable variation in these activities has been observed between people. No studies have reported on the correlation between human uptake of DBPs and water-zone mean estimates, but various studies found a good correlation between THM concentrations in exhaled breath and THM concentrations in water during showering and swimming. In general exposure assessment in epidemiological studies has been limited which complicates the interpretation. These findings have implications for epidemiological studies, particularly with reference to Berkson and classical error type models, study power, attenuation and precision of health-risk estimates and study efficiency. Recommendations are made for further areas of study.

Epidemiology, risk factors, and pathogenesis of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a fatal cancer of the biliary epithelium, arising either within the liver (intrahepatic, ICC) or in the extrahepatic bile ducts (extrahepatic ECC). Globally, CCA is the second most common primary hepatic malignancy. Several recent epidemiological studies have shown that the incidence and mortality rates of ICC are increasing. This review of the literature on the international epidemiological rates of CCA, both intra- and extrahepatic, explores possible explanations for the trends found. The possible role of epidemiological artifact in the findings is discussed and the known risk factors for CCA are summarized. These include primary sclerosing cholangitis, liver fluke infestation, congenital fibropolycystic liver, bile duct adenomas, and biliary papillomatosis, hepatolithiasis, chemical carcinogens such as nitrosamines, Thorotrast, chronic viral hepatitis, cirrhosis, chronic non-alcoholic liver disease and obesity. Potential pathways involved in the molecular pathogenesis of CCA are also summarized.

Modulation of transcription factor NF-kappa B binding activity by oxidation-reduction in vitro.

NF-kappa B is a widely used regulator of inducible and tissue-specific gene control. In the cytosol, when complexed to an inhibitory molecule, I kappa B, NF-kappa B is in an inactive form and cannot bind DNA. Activation of cells with appropriate stimuli results in the dissociation of NF-kappa B from I kappa B and its translocation to the nucleus as an active binding protein. We now demonstrate that NF-kappa B binding in vitro can be inhibited by agents that modify free sulfhydryls. Binding is eliminated after treatment with N-ethylmaleimide, an alkylating agent, and diamide, an oxidizing agent. The diamide effect can be reversed by 2-mercaptoethanol. Further, 2-mercaptoethanol acts synergistically with deoxycholate plus Nonidet P-40 in converting inactive cytosolic NF-kappa B to an active DNA-binding form. It is therefore possible that modulation of the redox state of NF-kappa B could represent a post-translational control mechanism for this factor.

H2O2 sensing through oxidation of the Yap1 transcription factor.

The yeast transcription factor Yap1 activates expression of antioxidant genes in response to oxidative stress. Yap1 regulation involves nuclear accumulation, but the mechanism sensing the oxidative stress signal remains unknown. We provide biochemical and genetic evidence that upon H2O2 treatment, Yap1 is activated by oxidation and deactivated by enzymatic reduction with Yap1-controlled thioredoxins, thus providing a mechanism for autoregulation. Two cysteines essential for Yap1 oxidation are also essential for its activation by H2O2. The data are consistent with a model in which oxidation of Yap1 leads to disulfide bond formation with the resulting change of conformation masking recognition of the nuclear export signal by Crm1/Xpo1, thereby promoting nuclear accumulation of the protein. In sharp contrast to H2O2, diamide does not lead to the same Yap1 oxidized form and still activates mutants lacking cysteines essential for H2O2 activation, providing a molecular basis for differential activation of Yap1 by these oxidants. This is the first example of an H2O2-sensing mechanism in a eukaryote that exploits the oxidation of cysteines in order to respond rapidly to stress conditions.

A genetic investigation of the essential role of glutathione: mutations in the proline biosynthesis pathway are the only suppressors of glutathione auxotrophy in yeast.

In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of Deltagsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of Deltagsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that gamma-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a Deltagsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions.

Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for DNA binding and multimerization.

OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and positively regulates the expression of proteins important for the defense against hydrogen peroxide in Escherichia coli and Salmonella typhimurium. Using random mutagenesis, we isolated six nonrepressing OxyR mutants that were impaired in DNA binding. Five of the mutations causing the DNA binding defect mapped near the N-terminal helix-turn-helix motif conserved among the LysR family members, confirming that this region is a DNA binding domain in OxyR. The sixth nonrepressing mutant (with E-225 changed to K [E225K]) was found to be predominantly dimeric, in contrast to the tetrameric wild-type protein, suggesting that a C-terminal region defined by the E225K mutation is involved in multimerization.

Sp1 represses IL-2 receptor alpha chain gene expression.

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.

A proteome analysis of the cadmium response in Saccharomyces cerevisiae.

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.

Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation.

OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium. OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes. Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form. We also used a random mutagenesis approach to isolate eight constitutively active mutants. All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue. In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase. Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding.

Chlorination disinfection byproducts in water and their association with adverse reproductive outcomes: a review.

OBJECTIVES AND METHODS: Chlorination has been the major disinfectant process for domestic drinking water for many years. Concern about the potential health effects of the byproducts of chlorination has prompted the investigation of the possible association between exposure to these byproducts and incidence of human cancer, and more recently, with adverse reproductive outcomes. This paper evaluates both the toxicological and epidemiological data involving chlorination disinfection byproducts (DBPs) and adverse reproductive outcomes, and makes recommendations for future research. RESULTS AND CONCLUSIONS: Relatively few toxicological and epidemiological studies have been carried out examining the effects of DBPs on reproductive health outcomes. The main outcomes of interest so far have been low birth weight, preterm delivery, spontaneous abortions, stillbirth, and birth defects--in particular central nervous system, major cardiac defects, oral cleft, and respiratory, and neural tube defects. Various toxicological and epidemiological studies point towards an association between trihalomethanes (THMs), one of the main DBPs and marker for total DBP load, and (low) birth weight, although the evidence is not conclusive. Administered doses in toxicological studies have been high and even though epidemiological studies have mostly shown excess risks, these were often not significant and the assessment of exposure was often limited. Some studies have shown associations for DBPs and other outcomes such as spontaneous abortions, stillbirth and birth defects, and although the evidence for these associations is weaker it is gaining weight. There is no evidence for an association between THMs and preterm delivery. The main limitation of most studies so far has been the relatively crude methodology, in particular for assessment of exposure. RECOMMENDATIONS: Large, well designed epidemiological studies focusing on well defined end points taking into account relevant confounders and with particular emphasis on exposure characterisation are ideally needed to confirm or refute these preliminary findings. In practice, these studies may be impracticable, partly due to the cost involved, but this is an issue that can be put right--for example, by use of subsets of the population in the design of exposure models. The studies should also reflect differences of culture and water treatment in different parts of the world. To identify the specific components that may be of aetiological concern and hence to fit the most appropriate exposure model with which to investigate human exposure to chlorinated DBPs, further detailed toxicological assessments of the mixture of byproducts commonly found in drinking water are also needed.

A new antioxidant with alkyl hydroperoxide defense properties in yeast.

To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of a Saccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7. We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine. Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization. AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1- and Skn7-dependent manner. Similar to yeast Tsa1p, Ahp1p forms a disulfide-linked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function. Furthermore, in contrast to Tsa1p, which is specific for H2O2, Ahp1p is specific for organic peroxides. Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC. These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.

Spatial variation and temporal trends of testicular cancer in Great Britain.

Increases in testicular cancer incidence have been reported in several countries over a long period. Geographical variability has also been reported in some studies. We have investigated temporal trends and spatial variation of testicular cancer at ages 20-49 in Britain. Temporal trends in testicular cancer incidence were examined, 1974 to 1991 and in mortality, 1981-1997. Spatial variation in incidence was analysed across electoral wards, 1975 to 1991. We used Poisson regression to examine for regional and socio-economic effects and Bayesian mapping techniques to analyse small-area spatial variability. Incidence increased from 6.5 to 11.1 per 100 000 in men at ages 20-34, and from 5.6 to 9.7 per 100 000 in men at ages 35-49, while mortality declined by 50% in both age groups. Risks of testicular cancer varied across regional cancer registries, ranging from 0.79 (95% CI: 0.73-0.84) to 1.32 (95% CI: 1.25-1.38), and was higher in the most affluent compared with the most deprived areas. Analyses within 2 regions (one predominantly urban, the other predominantly rural) did not indicate any localized geographical clustering. The increasing incidence contrasted with a decreasing mortality over time in Great Britain, similar to that found in other countries. The higher risk in more affluent areas is not consistent with findings on social class at the individual level. The absence of any marked geographical variability at small area scale argues against a geographically varying environmental factor operating strongly in the aetiology of testicular cancer.