Persistent socioeconomic and racial and ethnic disparities in pathogen burden in the United States, 1999-2014.

The disproportionate burden of prevalent, persistent pathogens among disadvantaged groups may contribute to socioeconomic and racial/ethnic disparities in long-term health. We assessed if the social patterning of pathogen burden changed over 16 years in a U.S.-representative sample. Data came from 17 660 National Health and Nutrition Examination Survey participants. Pathogen burden was quantified by summing the number of positive serologies for cytomegalovirus, herpes simplex virus-1, HSV-2, human papillomavirus and Toxoplasma gondii and dividing by the number of pathogens tested, giving a percent-seropositive for each participant. We examined sex- and age-adjusted mean pathogen burdens from 1999-2014, stratified by race/ethnicity and SES (poverty-to-income ratio (PIR); educational attainment). Those with a PIR < 1.3 had a mean pathogen burden 1.4-1.8 times those with a PIR > 3.5, with no change over time. Educational disparities were even greater and showed some evidence of increasing over time, with the mean pathogen burden among those with less than a high school education approximately twice that of those who completed more than high school. Non-Hispanic Black, Mexican American and other Hispanic participants had a mean pathogen burden 1.3-1.9 times non-Hispanic Whites. We demonstrate that socioeconomic and racial/ethnic disparities in pathogen burden have persisted across 16 years, with little evidence that the gap is closing.

Distal antral gastropexy - a novel technique to prevent recurrence of giant hiatus hernia in selected cases - a cohort study.

INTRODUCTION: The recurrence rate of giant hiatus hernias (GHH) following repair is high (30%) and increases with the hernia size and previous revision surgery. The mechanism of recurrence is poorly understood. METHODS: This is a retrospective cohort study of all consecutive patients who underwent repair of giant hiatus hernia in a tertiary upper GI referral centre from November 2000 to November 2014. Patients who underwent redo surgery were identified and data on intra-operative findings and procedure performed at primary and redo surgery from their operation notes were collected. RESULTS: A total of 81 patients underwent primary repair of GHH over the 14 year study period. 10 (12.3%) had symptomatic/radiological recurrence of which 4 were found to have the distal stomach herniating into the chest despite having an intact intra-abdominal wrap/gastropexy. To prevent migration of the distal stomach into the chest, distal gastropexy - fixing the antrum to the anterior abdominal wall, was added to 'conventional' gastropexy in 5 subsequent cases, in whom the antrum was in the chest preoperatively. These cases have no evidence of recurrence at the end of 6 months follow up. CONCLUSION: Securing the antrum of stomach to the anterior abdominal wall may prevent migration of the distal stomach and other infracolic organs into the chest and thus reduce recurrence of some GHH where antrum had been in chest previously.

Is C-reactive protein a useful adjunct in selecting patients for emergency cholecystectomy by predicting severe/gangrenous cholecystitis?

UNLABELLED: Gangrenous cholecystitis is difficult to diagnose clinically and carries risk of morbidity and mortality if not treated urgently. Complex scoring systems exist to predict the condition. C reactive protein may be a single independent predictor as suggested in this small study. BACKGROUND: Gangrenous cholecystitis (GC) is a serious sequel of acute cholecystitis occurring in 2-30% patients and has a mortality of 0.2-0.5%. Urgent surgical intervention is important to reduce morbidity and mortality therefore it is important to identify patients with GC from non-severe cholecystitis. The aim of this study is to determine biochemical and radiological markers, which is associated with the development GC and the value of C-reactive protein (CRP) at different cut-offs in predicting GC. METHODS: This is an observational cohort study of all consecutive patients who presented with biliary symptoms to the emergency department in a large NHS Hospital in the UK, from January to December 2012. They had cholecystectomies performed either during index admission or electively at later date by a team of 4 upper gastrointestinal surgeons. The gangrenous nature of the gallbladder was determined by operative findings and/or histopathology results. Parameters including age, gender, albumin, jaundice, gallbladder wall thickness on ultrasound scan, highest preoperative white blood count (WBC) and CRP value, were examined for their predictive value. RESULTS: 141 patients presented with acute biliary problems. 22 underwent emergency cholecystectomy and 119 were discharged and called back for elective surgery. Of these, 16 were gangrenous (11%). Patients with GC were significantly older (p = 0.016), had significantly higher CRP (p < 0.001) and WBC (p = 0.001), significantly lower albumin levels (p < 0.001) and higher percentage with thick walled gallbladder (p < 0.001). We found that a CRP value of more than 200 mg/dL has a 50% positive predictive value and 100% negative predictive value in predicting gangrenous cholecystitis with 100% sensitivity and 87.9% specificity. CONCLUSIONS: In this study CRP on its own has been shown to have high predictive value in predicting GC, but larger studies are needed to validate this finding. Monitoring trend of CRP in patients with acute cholecystitis may help early diagnosis and decision for early surgical intervention.

MyD88 adaptor-like (Mal) functions in the epithelial barrier and contributes to intestinal integrity via protein kinase C.

MyD88 adapter-like (Mal)-deficient mice displayed increased susceptibility to oral but not intraperitoneal infection with Salmonella Typhimurium. Bone marrow chimeras demonstrated that mice with Mal-deficient non-hematopoietic cells were more susceptible to infection, indicating a role for Mal in non-myeloid cells. We observed perturbed barrier function in Mal(-/-) mice, as indicated by reduced electrical resistance and increased mucosa blood permeability following infection. Altered expression of occludin, Zonula occludens-1, and claudin-3 in intestinal epithelia from Mal(-/-) mice suggest that Mal regulates tight junction formation, which may in part contribute to intestinal integrity. Mal interacted with several protein kinase C (PKC) isoforms in a Caco-2 model of intestinal epithelia and inhibition of Mal or PKC increased permeability and bacterial invasion via a paracellular route, while a pan-PKC inhibitor increased susceptibility to oral infection in mice. Mal signaling is therefore beneficial to the integrity of the intestinal barrier during infection.

The bile acid receptor, TGR5, regulates basal and cholinergic-induced secretory responses in rat colon.

Bile acids (BA) are becoming increasingly appreciated as enteric hormones that regulate many aspects of intestinal physiology. The BA receptor, TGR5, has been recently shown to be expressed on enteric nerves and enterochromaffin cells (ECs), where its activation regulates small intestinal and colonic motility. Here, we show that TGR5 is also expressed on colonic epithelial cells and that its activation decreases basal secretory tone and inhibits cholinergic-induced secretory responses. Our data demonstrate a new role for TGR5 in regulating colonic fluid and electrolyte transport and suggest that the receptor represents a good therapeutic target for intestinal transport disorders.

Part 3. Assessment of genotoxicity and oxidative stress after exposure to diesel exhaust from U.S. 2007-compliant diesel engines: report on 1- and 3-month exposures in the ACES bioassay.

Human health hazards due to diesel exhaust (DE*) exposure have been associated with both solvent and combustion components. In the past, diesel engine exhaust components have been linked to increased mutagenicity in cultures of Salmonella typhimurium and mammalian cells (Tokiwa and Ohnishi 1986). In addition, DE has been shown to increase both the incidence of tumors and the induction of 8-hydroxy-deoxyguanosine adducts (8-OHdG) in ICR mice (Ichinose et al. 1997). Furthermore, DE is composed of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) and particulates. One such PAH, 3-nitrobenzanthrone (3-NBA), has been identified in DE and found in urban air. 3-NBA has been observed to induce micronucleus formation in DNA of human hepatoma cells (Lamy et al. 2004). The purpose of the current research, which is part of the Advanced Collaborative Emissions Study (ACES), a multidisciplinary program being carried out by the Health Effects Institute and the Coordinating Research Council, is to determine whether improvements in the engineering of heavy-duty diesel engines reduce the oxidative stress and genotoxic risk associated with exposure to DE components. To this end, the genotoxicity and oxidative stress of DE from an improved diesel engine was evaluated in bioassays of tissues from Wistar Han rats and C57BL/6 mice exposed to DE. Genotoxicity was measured as strand breaks using an alkaline-modified comet assay. To correlate possible DNA damage found by the comet assay, measurement of DNA-adduct formation was evaluated by a competitive enzyme-linked immunosorbent assay (ELISA) to determine the levels of free 8-OHdG found in the serum of the animals exposed to DE. 8-OHdG is a specific modified base indicating an oxidative type of DNA damage to DNA nucleotides. In addition, a thiobarbituric acid reactive substances (TBARS) assay was used to assess oxidative stress and damage in the form of lipid peroxidation in the hippocampus region of the brains of DE-exposed animals. Results from the comet assay showed no significant differences in rats between the control and exposed groups (P = 0.53, low exposure; P = 0.92, medium exposure; P = 0.77, high exposure) after 1 month of DE exposure. There were no differences between sexes in the responses of rats to these exposures. Likewise, there were no significant differences found after 3 months of exposure. Similarly, no significant differences were found between the mice exposed for 1 and 3 months to DE, nor were any differences found between sexes. Measurements of 8-OHdG in both mice and rats showed no significant difference among DE exposure groups (P = 0.46, mice; P = 0.86, rats). In mice, measured 8-OHdG was lower in the 3-month group than the 1-month group. In rats, the inverse was true. In mice, no significant differences in the levels of lipid peroxidation, as measured by TBARS, were found between the controls and DE exposure groups (P = 0.92), nor were there any differences between sexes. In rats, comparisons between the control and low-exposure groups approached significance, but no significant differences were found between the other DE exposure groups. Additionally, in rats, there were no significant differences between the 1- and 3-month DE exposure groups.

Evaluation of internet derived patient information.

INTRODUCTION: The internet is a widely used, powerful resource for patients to research medical conditions. There is an extensive amount of information available on the internet. It is important for patient information to be accurate and in an easily accessible format. This article aims to assess the quality of patient information on hydrocephalus and compares the findings with recent evaluations in other surgical specialties. METHODS: The term 'hydrocephalus' was searched for on the search engines http://www.google.com/, http://www.bing.com/ and http://www.yahoo.com/. The top 20 results of these searches were assessed using the University of Michigan consumer health website evaluation checklist. RESULTS: The quality of patient information websites on hydrocephalus is highly variable. Websites rarely provide sufficient authorship information, do not review their information regularly enough and only reference material occasionally. The background of the provider was found to influence the quality of the website, with academic and care providers creating the best websites. CONCLUSIONS: On comparing our findings with those of recent studies from other surgical specialties, it was found that there was often a conflict of interest between the background of the provider and the information supplied. It is recommended that clinicians personally research material for their patients to be able to guide them to suitable, accurate websites.

Peritonitis from an abdominal wall biloma: a unique reason to avoid prophylactic surgical drainage.

Prophylactic drainage of the peritoneal space after major surgery is widely practised despite evidence against its efficacy. We describe the case of a 56-year-old woman who underwent a converted cholecystectomy and whose correctly sited abdominal drain resulted in the formation of a biloma between the external and internal oblique musculature. Subsequent leakage from the biloma into the abdominal cavity presented as peritonitis days after surgery, necessitating an emergency laparotomy. This case represents the first reported description of an abdominal wall biloma as a complication of post-cholecystectomy abdominal drainage. The evidence surrounding prophylactic drainage is discussed.

Discrete-choice preference comparison between patients and doctors for the surgical management of oesophagogastric cancer.

BACKGROUND: Establishing preferences for surgery is paramount to preoperative patient counselling. This study aimed to prioritize and compare preferences of patients and doctors towards surgery for oesophagogastric cancer, to aid the counselling process. METHODS: A discrete-choice questionnaire containing hypothetical scenarios was designed to test patient preferences for six treatment attributes: mortality, morbidity, quality of life (QoL), cure rate, hospital type and surgeon's reputation. The survey was mailed to all patients who underwent oesophagogastric cancer resection from 2008 to 2009 at two teaching hospital sites. All doctors at these sites with previous experience in counselling patients for cancer surgery were also identified and presented with the survey. Results were analysed using a random-effects probit regression model. Spearman correlation was used to compare participants' implicit choices from the discrete-choice scenarios (their true preferences) with their explicit choices from the direct ranking of preferences (their perceived preferences). RESULTS: Eighty-one patients and 90 doctors completed the survey. Some 15 per cent of patients and 31 per cent of doctors based their choices solely on QoL. In order of importance, patients based their implicit responses on QoL (ß = 1.19), cure rate (ß = 0.82), morbidity (ß = - 0.70), surgeon's reputation (ß = 0.60), mortality (ß = - 0.57) and hospital type (ß = 0.26). Doctors similarly indicated QoL (ß = 1.14) and hospital type (ß = 0.31) as highest and lowest preferences respectively, but placed far greater emphasis on mortality (ß = - 0.80) than morbidity (ß = - 0.35). Implicit and explicit preferences correlated only for morbidity and surgeon's reputation in the patient cohort. CONCLUSION: Clinicians may better meet patients' expectations and facilitate informed decision-making if QoL, cure rate and morbidity are emphasized foremost. A similar study employing preoperative patients is warranted for further clarification of preferences.

Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

BACKGROUND: Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function. METHODS: Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) colonic epithelial cells. Protein expression was measured by western blotting and intracellular Ca(2+) levels by Fura-2 fluorescence. KEY RESULTS: While acute (15 min) treatment of T(84) cells with a cholinergic G(q) PCR agonist, carbachol (CCh), rapidly stimulated Cl(-) secretion, subsequent CCh-induced responses were attenuated in a biphasic manner. The first phase was transient and resolved within 6 h but this was followed by a chronic phase of attenuated responsiveness that was sustained up to 48 h. CCh-pretreatment did not chronically alter responses to another G(q)PCR agonist, histamine, or to thapsigargin or forskolin which elevate intracellular Ca(2+) and cAMP, respectively. This chronically acting antisecretory mechanism is not shared by neurotransmitters that activate G(s)PCRs. Conditioned medium from CCh-pretreated cells mimicked its chronic antisecretory actions, suggesting involvement of an epithelial-derived soluble factor but further experimentation ruled out the involvement of epidermal growth factor receptor ligands. Acute CCh exposure did not chronically alter surface expression of muscarinic M(3) receptors but inhibited intracellular Ca(2+) mobilization upon subsequent agonist challenge. CONCLUSIONS & INFERENCES: These data reveal a novel, chronically acting, antisecretory mechanism that downregulates epithelial secretory capacity upon repeated G(q)PCR agonist exposure. This mechanism involves release of a soluble factor that uncouples receptor activation from downstream prosecretory signals.

Angelchik prosthesis with oesophageal adenocarcinoma: our surgical approach.

The Angelchik prosthesis is an incomplete doughnut-shaped device composed of silicone elastomer used in the treatment of gastro-oesophageal reflux disease (GORD). It is used to encircle the lower oesophagus at the gastro-oesophageal junction (GOJ). The ease of the operation led to the insertion of over 25,000 such prostheses world-wide. However, a variety of major complications including intractable dysphagia, prosthesis migration and gastric erosion required a quarter of these devices to be removed. Development of adenocarcinoma in patients with Angelchik prosthesis is a rare occurrence. This article describes two patients who developed adenocarcinoma above their prosthesis and whose cardio-oesophagectomy was technically challenging due to the formation of a dense inflammatory capsule around the prosthesis. Our surgical approach to curative oesophageal resection with the Angelchik prosthesis in situ is also discussed.

Hodgkin's disease of appendix: report of a case.

BACKGROUND: A number of cases of non Hodgkin's lymphoma of the appendix have been described, but Hodgkin's lymphoma is extremely rare. To our knowledge there are only two reports up to 1966 and none since then. METHOD: We report a case of a 65-year-old gentleman who was treated for suspected Crohn's disease. He failed to respond to medical treatment and underwent right haemicolectomy. The resected segment of bowel demonstrated classical Hodgkin's disease originating in the appendix. He recovered well from the operation and responded well to postoperative chemotherapy. CONCLUSION: Hodgkin's lymphoma of appendix is extremely rare. This case demonstrates the significance of repeated clinical evaluation of patients particularly in the absence of expected response to therapy.

3,4-Epoxy-1-butene, a reactive metabolite of 1,3-butadiene, induces somatic mutations in Xpc-null mice.

Xpc-null (Xpc-/-) mice, deficient in the global genome repair subpathway of nucleotide excision repair (NER-GGR), were exposed by intraperitoneal (i.p.) injection to a 300 mg/kg mutagenic dose of 3,4-epoxy-1-butene (EB), to investigate NER's potential role in repairing butadiene (BD) epoxide DNA lesions. Mutagenic sensitivity was assessed using the Hprt assay. Xpc-/- mice were significantly more sensitive to EB exposure, exhibiting an average 2.8-fold increase in Hprt mutant frequency (MF) relative to those of exposed Xpc+/+ (wild-type) mice. As a positive control for NER-GGR, additional mice were exposed by i.p. injection to a 150 mg/kg mutagenic dose of benzo[a]pyrene (B[a]P). The Xpc-/- mice had MFs 2.9-fold higher than those of exposed Xpc+/+ mice. These results suggest that NER-GGR plays a role in recognizing and repairing some of the DNA adducts formed following in vivo exposure to EB. Additional research is needed to examine the response of Xpc-/- mice, as well as other NER-deficient strains, to inhaled BD. Furthermore, it is likely that alternative DNA repair pathways also are involved in restoring genomic integrity compromised by BD-epoxide DNA damage. Collaborative studies are currently underway to address these critical issues.

Assessment of 1,3-butadiene exposure in polymer production workers using HPRT mutations in lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to make styrene-butadiene rubber, is a potent carcinogen in mice and a probable carcinogen, associated with leukemia, in humans. We have previously used HPRT mutation as a biomarker to evaluate exposures to BD in a monomer production plant. We now report on a study of 49 workers in a styrene-butadiene rubber plant in which we used the concentration of the BD metabolite 1,2-dihydroxy-4-(N-acetylcysteinyl-S)-butane (M1) in urine as a biomarker of exposure and the frequency of HPRT variant (mutant) lymphocytes (Vf) as a biomarker of effect. Workers were assigned to high- and low-exposure groups based on historical information about work areas and jobs. Personal exposure to BD for one work shift was measured using a passive badge dosimeter. Each participant provided a urine specimen and blood sample at the end of the work shift and completed a questionnaire providing information on lifestyle, health, and work activities. The average BD exposures in the high- and low-exposure groups were significantly different, even after excluding two extreme values, (high 1.48 ppm; low 0.15 ppm, p < 0.002). This study was done in 1994 and 1995 before the establishment, in 1996, of the new permissible exposure limit of 1 ppm. Both the mean M1 and the HPRT Vf were more than three times greater in the high-exposure group than in the low-exposure group (p < 0.0005). The three end points correlated with each other, with sample correlation coefficients between 0.4 and 0.6. The correlations among BD exposure and the biomarkers of internal exposure and genotoxicity suggest that occupational exposure to BD, in the range of 1-3 ppm, may be associated with adverse biological effects.

Biomarkers for assessing occupational exposures to 1,3-butadiene.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Assessment of butadiene exposure in synthetic rubber manufacturing workers in Texas using frequencies of hprt mutant lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to manufacture synthetic rubber, is a mutagen and carcinogen. Because past occupational exposures have been associated with an increased risk of leukemia, there has been a dramatic reduction in workplace exposure standards. The health benefits of these reduced levels of occupational exposure to BD will be difficult to evaluate using relatively insensitive traditional epidemiological studies; however, biomarkers can be used to determine whether there are genotoxic effects associated with recent exposures to BD. In past studies of BD-exposed workers in Southeast Texas, we observed an increase in the frequency of lymphocytes with mutations in a reporter gene, hprt. Frequencies of hprt mutant cells correlated with air levels of BD and with the concentration of a BD metabolite in urine. Average exposures to 1-3 parts per million (p.p.m.) of BD were associated with a threefold increase in hprt variant (mutant) frequencies (Vfs). We now report results from a follow-up study of workers in a synthetic rubber plant in Southeast Texas. Thirty-seven workers were evaluated on three occasions over a 2-week period for exposure to BD by the use of personal organic vapor monitors and by determining the concentration of a BD metabolite in urine. The frequency of hprt mutants was determined, by autoradiography, with lymphocyte samples collected 2 weeks after the final exposure measurement. Based on their work locations, the study participants were assigned to high-exposure (N=22) or low-exposure (N=15) groups. The BD exposure, +/-standard error, of the workers in the high-exposure group (1.65+/-0.52 p.p.m.) was significantly greater than the low-exposure group (0.07+/-0.03 p.p.m.; P<0.01). The frequency of hprt mutant lymphocytes was also significantly different in the two groups (high, 10.67+/-1.5 x 10(-6); low, 3.54+/-0.6 x 10(-6); P<0.001). The concentration of the urine metabolite was greater in the high-exposure group, but the difference was not significant. The correlation coefficient between hprt Vf and BD exposure levels was r=0.44 (CI(95), 0.11-0.69; P=0.011). This study reproduced the findings from a previous study at this plant. Although studies of butadiene-exposed workers in other countries have not detected an effect of exposure on frequencies of hprt mutant lymphocytes, we have repeatedly observed this result in our studies in Texas.

Human sensitivity to 1,3-butadiene: role of microsomal epoxide hydrolase polymorphisms.

1,3-Butadiene (BD) is a major commodity chemical used in the manufacture of synthetic rubber and various plastics and has been shown to be a potent animal carcinogen and a probable human carcinogen. The bioactivation of BD to reactive epoxides, and the balance between activation and detoxication of these reactive metabolites, is thought to play a critical role in the genotoxic and carcinogenic effects of BD. The detoxication of reactive BD metabolites involves enzymatic conjugation with glutathione by glutathione S-transferases (GSTs) and by hydrolysis, a reaction mediated by microsomal epoxide hydrolase (mEH). Since polymorphisms in genes of xenobiotic-metabolizing enzymes such as mEH may influence individual susceptibility to adverse health effects from BD exposure, we tested the hypothesis that the mEH Tyr113His polymorphism increases sensitivity to the genotoxic effects of BD in exposed workers. We used the autoradiographic hprt mutant lymphocyte assay as a biomarker of effect to identify genotoxicity associated with BD exposure in 49 workers from two styrene/butadiene polymer plants in Southeast Texas. Exposure to BD was assessed by collecting breathing zone air samples using passive badge dosimeters for three full 12 h work shifts 25, 20 and 14 days before blood was collected for genotyping and for the hprt assay. We genotyped the study participants for the Tyr113His polymorphism in the mEH gene and also for deletion polymorphisms in the glutathione S-transferase genes, GSTM1 and GSTT1, as potential biomarkers of susceptibility to BD. Our data indicate that the majority of the study subjects (67%) were exposed to very low levels of BD of <150 parts per billion (p.p.b.) time-weighted average (TWA). In some workers, however, we found levels of BD exposures that exceeded a TWA of 2000 p.p.b. Our data indicate a significant (P < 0.05) 2-fold increase in frequencies of hprt variant (mutant) lymphocytes (Vf) in workers exposed to >150 p.p.b. BD, compared with workers exposed to <150 p.p.b. There was no significant effect from individual GSTM1, GSTT1 or mEH genotypes in workers exposed to <150 p.p.b. BD. In workers exposed to >150 p.p.b., individuals with at least one polymorphic mEH His allele (His/His or His/Tyr genotypes) had a significant (P < 0.001) 3-fold increase in Vf (mean Vf x 10(-6) +/- SE = 13.25 +/- 1.78) compared with individuals with the Tyr/Tyr genotype (mean Vf x 10(-6) +/- SE = 4.02 +/- 0.72). There was no significant effect from individual GSTM1 or GSTT1 polymorphisms, but combined polymorphism analysis showed that the genetic damage was highest in individuals who had at least one mEH His allele and either the GSTM1 and/or GSTT1 null genotypes (hprt Vf = 14.19 +/- 2.30 x10(-6)). In contrast, this response was not observed in individuals exposed to levels of BD < 150 p.p.b. These results indicate that polymorphisms in the mEH gene may play a significant role in human sensitivity to the genotoxic effects of BD exposure, and that the hprt mutant lymphocyte assay can serve as a sensitive biomarker of genotoxicity for monitoring occupational exposure to BD in industrial settings. Additional investigations in larger populations of workers are needed to confirm our results and to characterize the possible role of additional mEH polymorphisms in the induction of genetic damage associated with occupational exposure to butadiene.

Point mutations induced by 1,2-epoxy-3-butene N1 deoxyinosine adducts.

The National Toxicology Program has recently classified 1,3-butadiene (BD) as a human carcinogen. BD is metabolized to the intermediates 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-dihydroxy-3,4-epoxybutane. All three metabolites have been implicated in producing specific types of DNA damage and as genotoxic agents in mice, rat, and human cells. This study has focused on EB-induced N1 deoxyinosine lesions that are formed by deamination of deoxyadenosine following reaction of the epoxide at the N(1) position. The R and S stereoisomers of this lesion were incorporated site-specifically within the context of an 11-mer oligodeoxynucleotide, incorporated into M13mp7L2 single-stranded DNA, and transfected into E. coli. Both stereoisomers modestly reduced plaque-forming ability, indicating that neither lesion presents a base modification that cannot be bypassed. The resulting plaques were assessed for point mutations using differential hybridization and DNA sequence analyses. The overall mutagenic spectrum revealed that the N1 adducts were highly mutagenic (approximately 90% per replication cycle), causing a predominance of A --> G transitions.

Molecular analysis of hprt mutant lymphocytes from 1, 3-butadiene-exposed workers.

1,3-Butadiene (BD) has been shown to be a potent animal carcinogen and a probable human carcinogen, yet the molecular mechanisms of BD genotoxicity and carcinogenicity still are not fully understood. Our hypothesis is that metabolites of BD induce specific structural changes in the human hprt gene like those observed in vitro in TK6 cells and in vivo in the mouse. Characteristic mutations in BD-exposed subjects can be identified and used as biomarkers for monitoring genotoxic effects associated with BD exposure. Molecular analysis of hprt mutant lymphocytes from BD-exposed workers and unexposed control subjects was carried out to identify changes in the structure of the hprt gene. A multiplex polymerase chain reaction (PCR) assay was used to detect exon deletions in 360 hprt mutant clones. We determined that exon deletions were significantly more frequent (P < 0.05) in BD-exposed workers (17.5%) than in control subjects (9.7%). Sequence analysis of hprt cDNA from 175 independent mutants indicated that the distribution of the types of mutations was different between the workers and the unexposed control subjects. There was a significant increase in -1 frameshift mutations in BD-exposed workers, predominantly in repeated DNA sequences, and single-base substitutions were decreased to 66% in the workers compared to 83% in the control subjects (P < 0.05). In addition to the spectral changes, hprt clonal assays revealed an elevation in mutant frequency in the lymphocytes of workers (N = 10) when compared with that in unexposed control subjects (N = 11; P < 0. 05). There also was a twofold increase of A:T --> T:A transversions in BD-exposed workers (16% in BD-exposed workers compared to 8% in controls, P = 0.25). Some of the BD-associated changes in mutational spectra observed in our study have the potential for application in monitoring genotoxic effects related to butadiene exposure.

Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality.

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.