Multicenter Evaluation of the Simplexa VZV Direct Assay for Detection of Varicella-Zoster Virus in Cerebrospinal Fluid and Lesion-Swab Specimens.

Varicella-zoster virus (VZV) is the etiologic agent of varicella (chickenpox) and herpes zoster (shingles) infections commonly involving skin, mucous membranes, and less frequently the central nervous system. Traditional methods for the laboratory diagnosis of these infections are time-consuming, labor-intensive, and often insensitive. As such, these tests are being replaced by more sensitive and rapid molecular methods. This study evaluated the performance of two different molecular assays, the Simplexa VZV Direct and Simplexa VZV Swab Direct, to detect VZV DNA in cerebrospinal fluid (CSF) and lesion-swab specimens, respectively. The Simplexa VZV Direct and Simplexa VZV Swab Direct assays were compared against individual composite reference methods that varied depending on the sample cohort examined. A total of 883 CSF and 452 cutaneous and mucocutaneous prospective, retrospective, and contrived specimens were evaluated in this multicenter study. The results of this study showed that the Simplexa assays demonstrated near perfect agreement (k = 0.98) compared to the composite reference methods for the detection of VZV in CSF and lesion swab specimens. A further comparison between the standard of care molecular assays employed at the site of specimen collection and the Simplexa assays demonstrated excellent agreement (k = 1.0). The Simplexa assays offer rapid and reliable alternatives for the detection of VZV in certain clinical specimens without the need for nucleic acid extraction.

A Randomized Clinical Trial of Standard versus Expanded Cultures to Diagnose Urinary Tract Infections in Women.

PURPOSE: We compared urinary tract infection (UTI) symptom resolution rates at 7-10 days in symptomatic women randomized to treatment based on standard urine culture (SUC) versus expanded quantitative urine culture (EQUC) results. MATERIALS AND METHODS: Women ≥18 years old who responded "yes" to "do you feel you have a UTI?" agreed to urethral catheterization and followup. Symptoms were assessed using the validated UTI Symptom Assessment (UTISA) questionnaire. Culture method was randomized 2:1 (SUC:EQUC); antibiotics were prescribed to women with positive cultures. The primary outcome, UTI symptom resolution, was determined 7-10 days following enrollment on all participants regardless of treatment. RESULTS: Demographic data were similar between groups. Of the SUC and EQUC groups 63% and 74% had positive cultures (p=0.10), respectively. Of participants with positive cultures 97% received antibiotics. Primary outcome data were provided by 215 of 225 participants (SUC 143 [95%], EQUC 72 [97%]). At the primary outcome assessment, 64% and 69% in the SUC and EQUC groups, respectively, reported UTI symptom resolution (p=0.46); UTISA scores improved from baseline in the EQUC arm compared to the SUC arm (p=0.04). In the subset of women predominated by non-Escherichia coli (76), there was a trend toward more symptom resolution in the EQUC arm (21%, p=0.08). CONCLUSIONS: Symptom resolution was similar for the overall population (E. coli and non-E. coli) of women treated for UTI symptoms based on SUC or EQUC. Although the sample size limits conclusions regarding the utility of EQUC in women with non-E. coli uropathogens, the detected trend indicates that this understudied clinical subset warrants further study.

Current Practice in Vitamin D Management in Allogeneic Hematopoietic Stem Cell Transplantation: A Survey by the Transplant Complications Working Party of the European Society for Blood and Marrow Transplantation.

Beyond its impact on bone health, numerous studies have investigated the immune-regulatory properties of vitamin D and shown how its deficiency can affect outcomes in allogeneic hematopoietic stem cell transplantation (HSCT), particularly in acute or chronic graft-versus-host disease. This survey, carried out by the Transplant Complications Working Party of the European Society for Blood and Marrow Transplantation (EBMT), describes the current clinical practice discrepancies across the EBMT HSCT programs. We therefore recommend the development of evidence-based guidelines to standardize evaluation criteria and to harmonize the management of vitamin D deficiency in patients undergoing allogeneic HSCT.

In vitro activity of oritavancin alone or in combination against vancomycin-susceptible and -resistant enterococci.

OBJECTIVES: The optimal treatment for serious infections due to Enterococcus spp. is unknown although combination antimicrobial therapy is often recommended for invasive infections to achieve bactericidal activity and improve clinical outcomes. Oritavancin is a novel lipoglycopeptide agent with in vitro activity against enterococci, including vancomycin-resistant VanA-type Enterococcus faecium. Data on its activity in combination with other antibacterials are limited. The objective of this study was to evaluate the activity of oritavancin alone and in combination with ceftriaxone, daptomycin, gentamicin, linezolid and rifampicin against vancomycin-susceptible and -resistant enterococci in in vitro time-kill analyses. METHODS: Five enterococcal strains were used for all experiments: three vancomycin-resistant VanA-type E. faecium clinical bloodstream isolates, vancomycin-resistant VanA-type E. faecium ATCC 700221 and vancomycin-susceptible Enterococcus faecalis ATCC 29212. Individual drugs were tested at », ½, 1, 2 and 4× MIC. Oritavancin combination experiments were performed with each agent at »× MIC. RESULTS: Daptomycin was the most active single agent and was bactericidal against all strains at 4× MIC, followed by oritavancin, which was bactericidal against all three clinical VRE strains at ≥2× MIC. In combination experiments at »× MIC, oritavancin was synergistic with gentamicin against strains not displaying high-level aminoglycoside resistance. No other synergy against VRE strains was observed in any experiment. Strain- and drug-dependent antagonism was observed for many combinations. CONCLUSIONS: These in vitro data do not support the routine use of combination therapy with oritavancin in the treatment of infections due to VRE.

Performance of ceftazidime/avibactam susceptibility testing methods against clinically relevant Gram-negative organisms.

OBJECTIVES: To ensure the accuracy of susceptibility testing methods for ceftazidime/avibactam. METHODS: The performances of the Etest (bioMérieux), 30/20 µg disc (Hardy diagnostics) and 10/4 µg disc (Mast Group) were evaluated against the reference broth microdilution (BMD) method for 102 clinically relevant Gram-negative organisms: 69 ceftazidime- and meropenem-resistant Klebsiella pneumoniae and 33 MDR non-K. pneumoniae. Essential and categorical agreement along with major and very major error rates were determined according to CLSI guidelines. RESULTS: A total of 78% of isolates were susceptible to ceftazidime/avibactam. None of the three methods met the defined equivalency threshold against all 102 organisms. The Etest performed the best, with categorical agreement of 95% and major errors of 6.3%. Against the 69 ceftazidime- and meropenem-resistant K. pneumoniae, only the Etest and the 10/4 µg disc met the equivalency threshold. None of the three methods met equivalency for the 33 MDR isolates. There were no very major errors observed in any analysis. These results were pooled with those from a previous study of 74 carbapenem-resistant Enterobacteriaceae and data from the ceftazidime/avibactam new drug application to define optimal 30/20 µg disc thresholds using the error-rate bound model-based approaches of the diffusion breakpoint estimation testing software. This analysis identified a susceptibility threshold of ≤19 mm as optimal. CONCLUSIONS: Our data indicate that the Etest is a suitable alternative to BMD for testing ceftazidime/avibactam against ceftazidime- and meropenem-resistant K. pneumoniae. The 30/20 µg discs overestimate resistance and may lead to the use of treatment regimens that are more toxic and less effective.

Diabetes-related major lower limb amputation incidence is strongly related to diabetic foot service provision and improves with enhancement of services: peer review of the South-West of England.

AIMS: To investigate the relationship between high diabetes-related lower limb amputation incidence and foot care services in the South-West region of England. METHODS: The introduction of 10 key elements of foot care service provision in one area of the South-West resulted in stabilization of foot ulcer incidence and sustained reduction in amputation incidence from 2007. Services introduced included administrative support, standardized general practice foot screening, improved community podiatry staffing, hospital multidisciplinary foot clinics, effective care pathways, availability of an orthotist and audit. Peer reviews of the region's diabetes foot care services were undertaken to assess delivery of these service provisions and compare this with major amputation incidence in other regions with data provided by Yorkshire and Humber Public Health Observatory Hospital Episode Statistics. Recommendations were made to improve service provision. In 2015 changes in service provision and amputation incidence were reviewed. RESULTS: Initial reviews in 2013 showed that the 3-year diabetes-related major amputation incidence correlated inversely with adequate delivery of diabetes foot care services (P=0.0024, adjusted R2 =0.51). Repeat reviews in 2015 found that two or more foot care service improvements were reported by six diabetes foot care providers, with improvement in outcomes. The negative relationship between major amputation incidence and service provision remained strong both in the period 2012-2015 and in the year 2015 only (P ≤0.0012, adjusted R2 =0.56, and P= 0.0005, R2 =0.62, respectively). CONCLUSIONS: Major diabetes-related lower limb amputation incidence is significantly inversely correlated with foot care services provision. Introduction of more effective service provision resulted in significant reductions in major amputation incidence within 2 years. Failure to improve unsatisfactory service provision resulted in continued high amputation incidence.

Nurses' communication with families in the intensive care unit - a literature review.

AIMS AND OBJECTIVES: This review examined the literature across 12 years that explored nurses' perceptions of their role when communicating with families in adult intensive care units (ICUs). The objectives were: (1) to describe how ICU nurses facilitated communication with families; (2) the perceived barriers to that communication; (3) strategies to improve their skills. BACKGROUND: Evidence demonstrates that effective communication by health care providers can improve families' understanding of their situation and decrease their psychological burden. The continuous presence of ICU nurses, combined with the use of effective empathic communication skills, can impact on families' ICU experience in a positive way. Nevertheless, research continues to demonstrate that communication with families in ICU is inadequate and of poor quality. SEARCH STRATEGIES AND DESIGN: Eight databases were systematically searched to identify peer reviewed studies published in English between 2002 and 2014. The results are presented via a thematic literature review. FINDINGS: Four major themes emerged from a synthesis of the review findings: 'Nurses as information and communication facilitators', 'Nurses as family support providers', 'Nurses' non-supportive behaviours' and 'Improving nurses' communication skills'. CONCLUSIONS: Most ICU nurses considered communicating with families a vital part of their role, and described supportive behaviours. However, they perceived significant barriers to effective communication; some as a result of active decisions on their part, and some beyond their control. These barriers often resulted in nurses believing that families received suboptimal information and support. Peer support and formal training were identified as key strategies to overcome inadequacies. RELEVANCE TO CLINICAL PRACTICE: This review summarizes nurses' perceptions of the means and barriers for communicating with families in ICUs. Intensive care nurses need skills and knowledge in how to communicate effectively with families. This skill can be learnt formally, or acquired from role modelling of more experienced peers.

Anemia increases the risk of renal cortical and medullary hypoxia during cardiopulmonary bypass.

INTRODUCTION: Anemia is an independent predictor of acute kidney injury (AKI) following cardiopulmonary bypass (CPB), possibly due to inadequate renal oxygen delivery. The objective of this study was to investigate the effects of CPB and anemia on tissue oxygen tension (pO2) and blood flow in the renal cortex and medulla. METHODS: Rats (n=6/group) underwent 1 hr of normothermic cardiopulmonary bypass (CPB), with target hemoglobin concentrations (Hb) of 10 g/dL (CPB) or 6.5 g/dL (anemia-CPB). Renal blood flow (RBF) and tissue PO2 were measured before, during and after 1 hr of CPB. To confirm the observed differences in renal cortical and medullary PO2, HIF-1α (ODD) luciferase mice were exposed to 8% O2 (hypoxia) and HIF-1α dependent luminescence was measured in the renal cortex and medulla (n=5). RESULTS: Renal tissue PO2 values decreased initially and returned towards baseline, however, values at the end of CPB. Anemia-CPB resulted in a significant increase in both renal cortical and medullary blood flow, PO2 remained significantly reduced throughout anemia-CPB. Renal medullary HIF-1α-dependent luminescence confirmed a greater degree of hypoxia in the renal medulla. DISCUSSION: During CPB, renal O2 delivery was transiently jeopardized, but recovered after 1 hr. Anemia-CPB resulted in a dramatic and sustained reduction in renal cortical and medullary PO2, which suggests an increased risk of renal hypoxic injury with anemia. CONCLUSION: The clear difference in the degree of hypoxia in the renal cortex and medulla may be useful in understanding the progress of medullary hypoxia during CPB with anemia and the potential development of AKI. Further studies should aim at identifying early markers of medullary hypoxia and potential agents that may decrease the work and O2 consumption in the renal medulla to reduce the risk of hypoxic damage during CPB and anemia.

Age similarities in matching pro- and contra-hedonic emotional strategies to everyday scenarios.

Older adults are motivated to maximize positive affect in the present. Young adults will purposely feel negative and high arousal emotions in order to achieve a goal. However, this type of contra-hedonic emotional alignment has not been extensively studied with older adults. We expected older adults are less likely than young adults to select high arousal and negative emotions within specific scenarios where those states could be useful. In two studies, participants selected the emotion they preferred in hypothetical problems that varied on the arousal and valence best suited for goal achievement. Young and older adults were equally likely to endorse affective strategies that matched both pro and contra-hedonic scenarios. While older adults may be generally motivated to avoid negative and high-arousing emotions, they are just as likely as young adults to indicate that these states could be helpful in certain situations.

Quantitative analysis and virulence phenotypes of atypical enteropathogenic Escherichia coli (EPEC) acquired from diarrheal stool samples from a Midwest US hospital.

Infectious diarrhea causes approximately 179 million illnesses annually in the US. Multiplex PCR assays for enteric pathogens detect enteropathogenic Escherichia coli (EPEC) in 12-29% of diarrheal stool samples from all age groups in developed nations. The aim of this study was to isolate and characterize EPEC from diarrhea samples identified as EPEC positive by BioFire Gastrointestinal Panel (GIP). EPEC is the second most common GIP-detected pathogen, equally present in sole and mixed infections peaking during summer months. EPEC bacterial load is higher in samples with additional pathogens. EPEC-GIP-positive stool samples were cultured on MacConkey II agar and analyzed by colony PCR for eaeA and bfpA to identify and classify EPEC isolates as typical (tEPEC) or atypical (aEPEC). EPEC were not recovered from the majority of stool samples with only 61 isolates obtained from 277 samples; most were aEPEC from adults. bfpA-mRNA was severely diminished in 3 of 4 bfpA-positive isolates. HeLa and SKCO-15 epithelial cells were infected with EPEC isolates and virulence-associated phenotypes, including adherence pattern, attachment level, pedestal formation, and tight junction disruption, were assessed. All aEPEC adherence patterns were represented with diffuse adherence predominating. Attachment rates of isolates adhering with defined adherence patterns were higher than tEPEC lacking bfpA (ΔbfpA). The majority of isolates formpedestals. All but one isolate initially increases but ultimately decreases transepithelial electrical resistance of SKCO-15 monolayers, similar to ΔbfpA. Most isolates severely disrupt occludin; ZO-1 disruption is variable. Most aEPEC isolates induce more robust virulence-phenotypes in vitro than ΔbfpA, but less than tEPEC-E2348/69.

Storage of strain-specific rat blood limits cerebral tissue oxygen delivery during acute fluid resuscitation.

BACKGROUND: The effect of blood storage on tissue oxygen delivery has not been clearly defined. Some studies demonstrate reduced microvascular oxygen delivery, whereas others do not. We hypothesize that storage of rat blood will limit its ability to deliver oxygen to cerebral tissue. METHODS: Anaesthetized rats underwent haemorrhage (18 ml kg(-1)) and resuscitation with an equivalent amount of fresh or 7 day stored strain-specific whole blood. Arterial blood gases, co-oximetry, red cell counts and indices, and blood smears were performed. Hippocampal tissue oxygen tension (PBr(O2)), regional cerebral blood flow (rCBF), and mean arterial pressure (MAP) were measured before and for 60 min after resuscitation (n=6). Data [mean (SD)] were analysed by anova. RESULTS: After 7 days, there was a significant reduction in pH, Pa(O2), an increase in Pa(CO2), but no detectable plasma haemoglobin in stored rat blood. Stored red blood cell morphology demonstrated marked echinocytosis, but no haemolysis in vitro. MAP and PBr(O2) in both groups decreased after haemorrhage. Resuscitation with stored blood returned MAP [92 (SD 16) mm Hg] and PBr(O2) [3.2 (0.7) kPa] to baseline, whereas rCBF remained stable [1.2 (0.1)]. Resuscitation with fresh blood returned MAP to baseline [105 (16) mm Hg] whereas both PBr(O2) [5.6 (1.5) kPa] and rCBF [1.9 (0.4)] increased significantly (P<0.05 for both, relative to baseline and stored blood group). There was no evidence of haemolysis in vivo. CONCLUSIONS: Although resuscitation with stored blood restored cerebral oxygen delivery to baseline, fresh blood produced a greater increase in both PBr(O2) and rCBF. These data support the hypothesis that storage limits the ability of RBC to deliver oxygen to brain tissue.

Impaired urinary concentration after vasopressin and its gradual correction in hypothalamic diabetes insipidus.

This study utilized rates with hereditary hypothalamic diabetes insipidus (D.I.) in order to explore possible mechanisms which prevent full urinary concentration after acute administration of vasopressin in hypothalamic D.I. and which correct this concentrating defect with prolonged therapy.IT WAS FOUND: (a) that the concentrating defect persisted even when the urinary osmolal excretion of D. I. rats was reduced to that of normal animals; (b) that the defect was not corrected more rapidly if larger doses of vasopressin were given; (c) that it persisted even when the D.I. rats were deprived of drinking water after vasopressin was given; (d) that there was osmotic equilibration between urine and renal papilla at a time when the concentrating defect was still evident; and (e) that the correction of the defect was associated with progressive and significant rise of the papillary osmolality. These studies appear to rule out osmotic diuresis, accumulation of exogenous vasopressin, persistent primary polydipsia, or delay in the induction of membrane permeability as causes for the concentrating defect. Rather, subnormal osmolality of the renal papilla, which can be corrected only gradually, accounts for the initial concentrating defect and the long time required for its correction. Reduction of water content and increase of urea content are primarily responsible for restoration of papillary osmolality to normal.

On the role of antidiuretic hormone in the inhibition of acute water diuresis in adrenal insufficiency and the effects of gluco- and mineralocorticoids in reversing the inhibition.

In order to determine whether or not antidiuretic hormone (ADH) is essential to the inhibition of an acute water diuresis in adrenal insufficiency, the response to oral water loads was tested in rats with hereditary hypothalamic diabetes insipidus (DI) which lack ADH. It was found that 60 min after water loads of 3 or 5% of body weight urine flow was significantly lower and urine osmolality significantly higher in adrenalectomized DI rats than in the same DI rats before removal of their adrenal glands. The efficacy of gluco- and mineralocorticoids in reversing the inhibition was then determined in the same adrenalectomized DI rats. Prednisolone alone, administered either acutely or chronically, restored the response in urine flow to that seen in the same rats before adrenalectomy, but failed to correct the defect in urinary dilution. Aldosterone when given alone tended to correct the diluting ability but not the response in urine flow. When these two adrenal cortical hormones were given simultaneously, both the urine flow and urine osmolality were nearly identical to what they had been in the same DI rats before adrenalectomy. These studies strongly suggest (a) that ADH is not essential to the inhibition of an acute water diuresis in adrenal insufficiency, although it may abet the inhibition in individuals without diabetes insipidus, which can elaborate ADH; and (b) that both gluco- and mineralocorticoids are required in adrenal insufficiency in order to fully restore the water diuresis as judged by the dual criteria of urine flow and urine osmolality.

Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system.

Little is known regarding the location of cholinergic muscarinic receptor 1 (M1r) in the ENS, even though physiological data suggest that M1rs are central to cholinergic neurotransmission. This study localised M1rs in the ENS of the guinea pig ileum and human colon using fluorescence immunohistochemistry and RT-PCR in human colon. Double labelling using antibodies against neurochemical markers was used to identify neuron subytpes bearing M1r. M1r immunoreactivity (IR) was present on neurons in the myenteric and submucosal ganglia. The two antibodies gave similar M1r-IR patterns and M1r-IR was abolished upon antibody preabsorption. M1r-IR was present on cholinergic and nNOS-IR nerve cell bodies in both guinea pig and human myenteric neurons. Presynaptic M1r-IR was present on NOS-IR and VAChT-IR nerve fibres in the circular muscle in the human colon. In the submucosal ganglia, M1r-IR was present on a population of neurons that contained cChAT-IR, but did not contain NPY-IR or calretinin-IR. M1r-IR was present on endothelial cells of blood vessels in the submucosal plexus. The localisation of M1r-IR in the guinea pig and human ENS shown in this study agrees with physiological studies. M1r-IR in cholinergic and nitrergic neurons and nerve fibres indicate that M1rs have a role in both cholinergic and nitrergic transmission. M1r-IR present in submucosal neurons suggests a role in mediating acetylcholine's effect on submucosal sensory and secretomotor/vasodilator neurons. M1r-IR present on blood vessel endothelial cells suggests that M1rs may also mediate acetylcholine's direct effect on vasoactivation.

Hallucinogen-like actions of 5-methoxy-N,N-diisopropyltryptamine in mice and rats.

Few studies have examined the effects of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in vivo. In these studies, 5-MeO-DIPT was tested in a drug-elicited head twitch assay in mice where it was compared to the structurally similar hallucinogen N,N-dimethyltryptamine (N,N-DMT) and challenged with the selective serotonin (5-HT)2A antagonist M100907, and in a lysergic acid diethylamide (LSD) discrimination assay in rats where its subjective effects were challenged with M100907 or the 5-HT 1A selective antagonist WAY-100635. Finally, the affinity of 5-MeO-DIPT for three distinct 5-HT receptors was determined in rat brain. 5-MeO-DIPT, but not N,N-DMT, induced the head twitch responses in the mouse, and this effect was potently antagonized by prior administration of M100907. In rats trained with LSD as a discriminative stimulus, there was an intermediate degree (75%) of generalization to 5-MeO-DIPT and a dose-dependent suppression of response rates. These interoceptive effects were abolished by M100907, but were not significantly attenuated by WAY-100635. Finally, 5-MeO-DIPT had micromolar affinity for 5-HT 2A and 5-HT 2C receptors, but much higher affinity for 5-HT 1A receptors. 5-MeO-DIPT is thus effective in two rodent models of 5-HT2 agonist activity, and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of this compound, coupled with the lack of significant antagonist effects of WAY-100635, strongly suggests that the 5-HT 2A receptor is an important site of action for 5-MeO-DIPT, despite its apparent in vitro selectivity for the 5-HT 1A receptor.

Human lung carcinogen-DNA adduct levels mediated by genetic polymorphisms in vivo.

BACKGROUND: Cancer risk from exposure to tobacco smoke varies widely from person to person, depending in part on the status of particular genes and acquired susceptibilities. Certain genes determine how cells activate and detoxify carcinogens. Activated carcinogen metabolites may bind to DNA and form DNA adducts (e.g., 7-methyl-2'-deoxyguanosine-3'-monophosphate [7-methyl-dGMP] and polycyclic aromatic hydrocarbons-dGMP [PAHs-dGMP]), many of which can induce genetic mutations. Thus, if individuals have an increased capacity to activate carcinogens, they might form more carcinogen-DNA adducts and subsequently have an increased risk of cancer. PURPOSE: Using DNA-adduct detection methods specific for 7-methyl-dGMP and PAH-dGMP, we sought to determine whether an inherited genetic susceptibility to cancer associated with certain carcinogen-metabolizing and detoxifying genes (e.g., cytochrome P450 and glutathione S-transferase) is related to DNA adduct formation in lung tissue. METHODS: Human lung tissues were collected randomly from 90 autopsy donors who were free of cancer. Levels of 7-methyl-dGMP, a metabolic product of N-nitrosamines, and PAH-dGMP adducts were determined in lung tissue specimens by use of micropreparative DNA purification steps combined with a 32P-postlabeling assay. Genetic polymorphisms (the presence of different genes and/or alleles) were determined for the cytochrome P450 genes, CYP2D6, CYP2E1, and CYP1A1, as well as for glutathione S-transferase M1 (GSTM1). Statistical differences among adduct levels for the study variables, including genotypes, were assessed by the two-sided Student's t test or the Mann-Whitney U test. RESULTS: Higher 7-methyl-dGMP adduct levels were associated with CYP2D6 genotypes (P = .01), consistent with the reports of the increased risk of lung cancer associated with this genotype. Higher adduct levels were also associated with CYP2E1 minor alleles (P = .05). In both cases, the association was attributed mostly to individuals with low serum cotinine levels (P = .004 and P = .05, respectively), suggesting that the effect of the genotypes is mostly in nonsmokers exposed to either passive tobacco smoke or to N-nitrosamine exposures other than tobacco smoke or to N-nitrosamine exposures other than tobacco smoke. Separately, the presence of PAH-dGMP adducts was associated with the GSTM1 null genotype (absence of the gene) (odds ratio = 8.6; 95% confidence interval = 1.03-100). CONCLUSIONS: This study finds that the levels of two different carcinogen-DNA adducts vary in lung tissue (an important target tissue) in association with three separate genetic polymorphisms (i.e., CYP2D6, CYP2E1, and GSTM1). CYP2D6 and CYP2E1 genotypes are associated with higher 7-methyl-dGMP levels, while the GSTM1 null genotype is associated with higher numbers of PAH-dGMP adducts. These findings suggest that genetic polymorphisms are predictive of carcinogen-DNA adduct levels and would thus be predictive of an individual's lifetime response to carcinogen exposure.

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes.

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.

Cytochrome P4501A1 and glutathione S-transferase (M1) genetic polymorphisms and postmenopausal breast cancer risk.

Polycyclic aromatic hydrocarbons, possible human breast carcinogens, are metabolized by cytochrome P4501A1 (CYP1A1) and glutathione S-transferase (GSTM1). A CYP1A1 polymorphism (isoleucine to valine substitution in exon 7) or the null allele for GSTM1 may affect the mutagenic potential of polycyclic aromatic hydrocarbons. We examined polymorphisms in GSTM1 and CYP1A1 in relation to breast cancer risk. Included were 216 postmenopausal Caucasian women with incident breast cancer and 282 community controls. DNA analyses suggested no increased breast cancer risk with the null GSTM1 genotype [odds ratio (OR) = 1.10; CI, 0.73-1.64], although there was some indication that the null genotype was associated with risk among the youngest postmenopausal women (OR = 2.44; CI, 0.89-6.64). Slightly elevated risk was associated with the CYP1A1 polymorphism (OR = 1.61; CI, 0.94-2.75) and was highest for those who smoked up to 29 pack-years (OR = 5.22; CI, 1.16-23.56). Statistical power to detect an effect may be limited by small numbers, and larger sample sizes would be required to corroborate these suggestive findings.