Polycyclic Aromatic Hydrocarbons and Pancreatic Cancer: An Analysis of the Blood Biomarker, r-1,t-2,3,c-4-Tetrahydroxy-1,2,3,4-tetrahydrophenanthrene and Selected Metabolism Gene SNPs.

Exposure to polycyclic aromatic hydrocarbons (PAHs), byproducts of incomplete combustion, and their effects on the development of cancer are still being evaluated. Recent studies have analyzed the relationship between PAHs and tobacco or dietary intake in the form of processed foods and smoked/well-done meats. This study aims to assess the association of a blood biomarker and metabolite of PAHs, r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), dietary intake, selected metabolism SNPs, and pancreatic cancer. Demographics, food-frequency data, SNPs, treatment history, and levels of PheT in plasma were determined from 400 participants (202 cases and 198 controls) and evaluated based on pancreatic adenocarcinoma diagnosis. Demographic and dietary variables were selected based on previously published literature indicating association with pancreatic cancer. A multiple regression model combined the significant demographic and food items with SNPs. Final multivariate logistic regression significant factors (p-value < 0.05) associated with pancreatic cancer included: Type 2 Diabetes [OR = 6.26 (95% CI = 2.83, 14.46)], PheT [1.03 (1.02, 1.05)], very well-done red meat [0.90 (0.83, 0.96)], fruit/vegetable servings [1.35 (1.06, 1.73)], recessive (rs12203582) [4.11 (1.77, 9.91)], recessive (rs56679) [0.2 (0.06, 0.85)], overdominant (rs3784605) [3.14 (1.69, 6.01)], and overdominant (rs721430) [0.39 (0.19, 0.76)]. Of note, by design, the level of smoking did not differ between our cases and controls. This study does not provide strong evidence that PheT is a biomarker of pancreatic cancer susceptibility independent of dietary intake and select metabolism SNPs among a nonsmoking population.

School Gardens Enhance Academic Performance and Dietary Outcomes in Children.

BACKGROUND: Schools face increasing demands to provide education on healthy living and improve core academic performance. Although these appear to be competing concerns, they may interact beneficially. This article focuses on school garden programs and their effects on students' academic and dietary outcomes. METHODS: Database searches in CABI, Web of Science, Web of Knowledge, PubMed, Education Full Text, Education Resources Information Center (ERIC), and PsychINFO were conducted through May 2013 for peer-reviewed literature related to school-day garden interventions with measures of dietary and/or academic outcomes. RESULTS: Among 12 identified garden studies with dietary measures, all showed increases/improvements in predictors of fruit and vegetable (FV) consumption. Seven of these also included self-reported FV intake with 5 showing an increase and 2 showing no change. Four additional interventions that included a garden component measured academic outcomes; of these, 2 showed improvements in science achievement and 1 measured and showed improvements in math scores. CONCLUSIONS: This small set of studies offers evidence that garden-based learning does not negatively impact academic performance or FV consumption and may favorably impact both. Additional studies with more robust experimental designs and outcome measures are necessary to understand the effects of experiential garden-based learning on children's academic and dietary outcomes.

Factors affecting fruit and vegetable school lunch waste in Wisconsin elementary schools participating in Farm to School programmes.

OBJECTIVE: To examine characteristics potentially associated with school lunch fruit and vegetable waste, both overall and pre/post implementation of the Healthy, Hunger-Free Kids Act. DESIGN: Multi-year (2010-2013) cross-sectional study using pre- and post-meal digital photographs of students' school lunch trays to estimate fruit and vegetable availability and consumption. Fruit and vegetable items were categorized for factors suspected to impact waste: prior farm to school years, placement (main menu, salad bar), procurement (local, conventional), preparation (cooked, raw) and meal component (entrée, side, topping). Analyses to assess within-category differences in waste volume were performed using a Tobit model. SETTING: Wisconsin elementary schools participating in farm to school programmes, USA. SUBJECTS: Children in third to fifth grade. RESULTS: Many within-factor differences were detected overall and/or across time. Cooked fruits were wasted less than raw, while cooked vegetables were wasted more than raw. Where identified, locally sourced items were wasted more than conventionally sourced (+0·1 cups, P<0·0001) and salad bar items more than main menu items (+0·01 cups, P<0·0001). Increasing prior farm to school years decreased waste (-0·02 cups, P<0·0001). Items previously tried were wasted at the same volume whether reported as liked or not. New school lunch meal pattern requirement implementation did not uniformly impact fruit and vegetable waste across all categories and there was no change in waste for seven of fifteen assessed categories. CONCLUSIONS: Many factors impact elementary students' school lunch waste. These factors may be helpful for school food-service authorities to consider when planning school menus.

Fruits and vegetables displace, but do not decrease, total energy in school lunches.

BACKGROUND: The high overweight and obesity prevalence among US children is a well-established public health concern. Diet is known to play a causal role in obesity. Increasing fruit and vegetable (FV) consumption to recommended levels is proposed to help reduce obesity, because their bulk and low energy density are believed to reduce energy-dense food consumption (volume displacement hypothesis). This study tests this hypothesis at the lunch meal among upper-elementary students participating in a Farm to School (F2S) program. METHODS: Digital photographs of students' school lunch trays were visually analyzed to identify the food items and amounts that were present and consumed before and after the meal. Using the USDA Nutrient Database, total and FV-only energy were calculated for each tray. Analysis of total- and non-FV energy intake was performed according to (1) levels of FV energy intake, (2) FV energy density, and (3) previous years of Farm to School programming. RESULTS: Higher intake of FV energy displaced non-FV energy, but total energy did not decrease across FV energy intake groups. High-FV-energy-density trays showed lower non-FV energy intake than low-FV-energy-density trays (470±179 vs. 534±219 kcal; p<0.0001). Trays from schools with more previous years of F2S programming decreased total and non-FV energy intake from school lunches (p for trend<0.0001, both). CONCLUSIONS: Increased FV consumption reduces non-FV energy intake, but does not reduce total energy intake. Therefore, this study does not support the volume displacement hypothesis and suggests calorie displacement instead.

Farm to elementary school programming increases access to fruits and vegetables and increases their consumption among those with low intake.

OBJECTIVE: To assess the effectiveness of Wisconsin Farm to School (F2S) programs in increasing students' fruit and vegetable (FV) intake. DESIGN: Quasi-experimental baseline and follow-up assessments: knowledge and attitudes survey, food frequency questionnaire (FFQ), and lunch tray photo observation. SETTING: Wisconsin elementary schools: 1 urban and 8 rural. PARTICIPANTS: Children, grades 3-5 (n = 1,117; 53% male, 19% non-Caucasian). INTERVENTION(S): Farm to School programming ranging from Harvest of the Month alone to comprehensive, including school garden, locally sourced produce in school meals, and classroom lessons. MAIN OUTCOME MEASURES: Knowledge, attitudes, exposure, liking, willingness; FFQ-derived (total), and photo-derived school lunch FV intake. ANALYSIS: t tests and mixed modeling to assess baseline differences and academic-year change. RESULTS: Higher willingness to try FV (+1%; P < .001) and knowledge of nutrition/agriculture (+1%; P < .001) (n = 888), and lunch FV availability (+6% to 17%; P ≤ .001) (n = 4,451 trays), both with increasing prior F2S program exposure and across the year. There was no effect on overall dietary patterns (FFQ; n = 305) but FV consumption increased among those with the lowest intakes (FFQ, baseline very low fruit intake, +135%, P < .001; photos: percentage of trays with no FV consumption for continuing programs decreased 3% to 10%, P ≤ .05). CONCLUSIONS AND IMPLICATIONS: Farm to School programming improved mediators of FV consumption and decreased the proportion of children with unfavorable FV behaviors at school lunch. Longer-term data are needed to further assess F2S programs.

High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitrosamine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in smokers.

We developed and applied high throughput liquid and gas chromatography-tandem mass spectrometry (LC-MS/MS and GC-MS/MS) methods for the cigarette smoking-associated biomarkers 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), which are urinary metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the polycyclic aromatic hydrocarbon phenanthrene. NNAL and PheT levels have been linked to lung cancer in previous studies of smokers. Confirmation of these relationships will require further molecular epidemiology studies, necessitating improved methodology applicable to large numbers of small urine samples. Furthermore, NNAL is excreted in urine either unconjugated or as an N- or O-glucuronide, but little data are available on the amounts of each in urine. For the high throughput analysis of NNAL, 3 aliquots were processed from each urine sample, one for the analysis of free NNAL, one for free NNAL plus NNAL-N-Gluc, and one for total NNAL (the sum of free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc). Ninety-six well plate technology was used for sample enrichment by supported liquid extraction plates, mixed mode reverse-phase/cation exchange solid-phase extraction, and LC-MS/MS analysis. For the analysis of PheT, the urine samples were cleaned up by solid-phase extraction on styrene-divinylbenzene sorbent, silylated, and analyzed by GC-MS/MS, both in 96-well format. The methods were validated analytically with respect to accuracy and precision, and applied in an ongoing molecular epidemiology study of smokers. The amount of total NNAL in smokers' urine was (mean ± SD) 1.65 ± 2.13 pmol/mL (N = 2641). Free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc represented (mean ± SD) 31 ± 11%, 22 ± 14%, and 48 ± 15% of total NNAL, respectively. The amount of PheT in smokers' urine was (mean ± SD) 1.43 ± 2.16 pmol/mL (N = 2613). The methodology described here should be widely applicable in future studies of tobacco use and cancer.

A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B.

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.

Metabolites of a tobacco-specific lung carcinogen in children exposed to secondhand or thirdhand tobacco smoke in their homes.

BACKGROUND: People exposed to secondhand tobacco smoke (SHS) inhale the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which is metabolized to NNAL and its glucuroniders. These urinary metabolites, termed total NNAL, can be quantified. A related compound, iso-NNAL, has been proposed as a biomarker for exposure to smoke constituent residues on surfaces (thirdhand tobacco smoke). There is limited information in the literature on levels of total NNAL in children exposed to SHS. METHODS: We recruited 79 parent--child dyads from homes where the enrolled parent was a cigarette smoker and visited their homes. Parents were asked questions, home ambient air quality was evaluated, and children provided urine samples. Urine was analyzed for total NNAL, total cotinine, total nicotine, and iso-NNAL. RESULTS: Ninety percent of the children had detectable total NNAL in urine; total nicotine and total cotinine were also detected in most samples. There were significant positive relationships between biomarker levels and exposure of children in the home. Levels were highest in homes with no smoking restrictions. African American children had significantly higher levels than other children. iso-NNAL was not detected in any urine sample. CONCLUSIONS: There was nearly universal exposure of children to the lung carcinogen NNK, due mainly to exposure to SHS from adult smokers in their homes. IMPACT: Homes with adult smokers should adopt restrictions to protect their children from exposure to a potent lung carcinogen.

Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.

Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.

Temporal stability of urinary and plasma biomarkers of tobacco smoke exposure among cigarette smokers.

Intraindividual variability of measurements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), nicotine, cotinine, and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) over time is uncertain. From 70 habitual smokers' plasma and urine sampled bimonthly for a year we analysed plasma for NNAL, cotinine and PheT, and urine for NNAL, cotinine and nicotine. We estimated the intraclass correlation coefficients (rho(I)) for each measurement. Plasma and creatinine-corrected urinary NNAL were stable (rho(I) > or =70%); plasma PheT and plasma and urinary total cotinine were fairly stable (rho(I) > or =50%), but urinary nicotine rho(I) approximately 40% was not. Except for nicotine, single measurements from plasma or urine adequately represent individual mean exposure over time.

A familial mutation renders atrial natriuretic Peptide resistant to proteolytic degradation.

A heterozygous frameshift mutation causing a 12-amino acid extension to the C terminus of atrial natriuretic peptide (ANP) was recently genetically linked to patients with familial atrial fibrillation (Hodgson-Zingman, D. M., Karst, M. L., Zingman, L. V., Heublein, D. M., Darbar, D., Herron, K. J., Ballew, J. D., de Andrade, M., Burnett, J. C., Jr., and Olson, T. M. (2008) N. Engl. J. Med. 359, 158-165). The frameshift product (fsANP), but not wild-type ANP (wtANP), was elevated in the serum of affected patients, but the molecular basis for the elevated peptide concentrations was not determined. Here, we measured the ability of fsANP to interact with natriuretic peptide receptors and to be proteolytically degraded. fsANP and wtANP bound and activated human NPR-A and NPR-C similarly, whereas fsANP had a slightly increased efficacy for human NPR-B. Proteolytic susceptibility was addressed with novel bioassays that measure the time required for kidney membranes or purified neutral endopeptidase to abolish ANP-dependent activation of NPR-A. The half-life of fsANP was markedly greater than that of wtANP in both assays. Additional membrane proteolysis studies indicated that wtANP and fsANP are preferentially degraded by neutral endopeptidase and serine peptidases, respectively. These data indicate that the familial ANP mutation associated with atrial fibrillation has only minor effects on natriuretic peptide receptor interactions but markedly modifies peptide proteolysis.

Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications.

Natriuretic peptides are a family of three structurally related hormone/ paracrine factors. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are secreted from the cardiac atria and ventricles, respectively. ANP signals in an endocrine and paracrine manner to decrease blood pressure and cardiac hypertrophy. BNP acts locally to reduce ventricular fibrosis. C-type natriuretic peptide (CNP) primarily stimulates long bone growth but likely serves unappreciated functions as well. ANP and BNP activate the transmembrane guanylyl cyclase, natriuretic peptide receptor-A (NPR-A). CNP activates a related cyclase, natriuretic peptide receptor-B (NPR-B). Both receptors catalyze the synthesis of cGMP, which mediates most known effects of natriuretic peptides. A third natriuretic peptide receptor, natriuretic peptide receptor-C (NPR-C), clears natriuretic peptides from the circulation through receptor-mediated internalization and degradation. However, a signaling function for the receptor has been suggested as well. Targeted disruptions of the genes encoding all natriuretic peptides and their receptors have been generated in mice, which display unique physiologies. A few mutations in these proteins have been reported in humans. Synthetic analogs of ANP (anaritide and carperitide) and BNP (nesiritide) have been investigated as potential therapies for the treatment of decompensated heart failure and other diseases. Anaritide and nesiritide are approved for use in acute decompensated heart failure, but recent studies have cast doubt on their safety and effectiveness. New clinical trials are examining the effect of nesiritide and novel peptides, like CD-NP, on these critical parameters. In this review, the history, structure, function, and clinical applications of natriuretic peptides and their receptors are discussed.

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab -/- mice.

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.

Comparison of polymorphisms in genes involved in polycyclic aromatic hydrocarbon metabolism with urinary phenanthrene metabolite ratios in smokers.

The hypothesis that interindividual differences among smokers in the metabolism of polycyclic aromatic hydrocarbons (PAH) are related to lung cancer risk has been extensively investigated in the literature. These studies have compared lung cancer risk in groups of smokers with or without polymorphisms in genes involved in PAH metabolism. We believe that carcinogen metabolite phenotyping, involving the actual measurement of PAH metabolites, would be a better way to investigate differences in lung cancer risk. With this goal in mind, we have developed methods for quantifying phenanthrene metabolites in urine. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity. The urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) is a measure of metabolic activation, whereas phenanthrols (HOPhe) are a measure of detoxification. In this study, we quantified urinary PheT/HOPhe ratios in 346 smokers who were also genotyped for 11 polymorphisms in genes involved in PAH metabolism: CYP1A1MspI, CYP1A1I462V, CYP1B1R48G, CYP1B1A119S, CYP1B1L432V, CYP1B1N453S, EPHX1Y113H, EPHX1H139R, GSTP1I105V, GSTP1A114V, and GSTM1 null. The geometric mean molar PheT/3-HOPhe ratio was 4.08 (95% confidence interval, 3.79-4.39). Ten percent of the smokers had PheT/3-HOPhe ratios of > or =9.90. We found a significant association between the presence of the CYP1A1I462V polymorphism and high PheT/3-HOPhe ratios (P = 0.02). This effect was particularly strong in females and in combination with the GSTM1 null polymorphism. In contrast, the CYP1B1R48G and CYP1B1A119S polymorphisms were associated with significantly lower PheT/3-HOPhe ratios, particularly in Blacks. There were no consistent significant effects of any of the other polymorphisms on PheT/3-HOPhe ratios. The highest 10% of PheT/3-HOPhe ratios could not be predicted by the presence of any of the 11 polymorphisms individually or by certain combinations. The effects of the CYP1A1I462 polymorphism observed here, particularly in combination with GSTM1 null, are quite consistent with reports in the literature. However, the results of this study indicate that genotyping is not an effective way to predict PAH metabolism at least as represented by PheT/HOPhe ratios.

Combined analysis of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers' plasma.

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are widely accepted to be two important types of lung carcinogens in cigarette smoke. In this study, we have developed a method to estimate individual uptake of these compounds by quantifying r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in 1 mL of smokers' plasma. PheT and NNAL are biomarkers of PAH and NNK uptake, respectively. [D10]PheT and [pyridine-D4]NNAL were added to plasma as internal standards. The plasma was treated with beta-glucuronidase to release any conjugated PheT and NNAL. The analytes were enriched by solid-phase extraction on a mixed mode cation exchange cartridge and the PheT fraction was further purified by high-performance liquid chromatography. The appropriate fractions were analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry for PheT and liquid chromatography-electrospray ionization-mass spectrometry for NNAL. The method was sensitive (limits of quantitation: PheT, 13 fmol/mL; NNAL, 3 fmol/mL), accurate, and precise. Levels of PheT and NNAL in plasma from 16 smokers averaged 95 +/- 71 and 36 +/- 21 fmol/mL, respectively, which are approximately 1% to 2% of the amounts found in urine. This method should be useful in molecular epidemiology studies of carcinogen uptake and lung cancer in smokers.

Roles of endogenous ascorbate and glutathione in the cellular reduction and cytotoxicity of sulfamethoxazole-nitroso.

Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

Longitudinal study of urinary phenanthrene metabolite ratios: effect of smoking on the diol epoxide pathway.

We have proposed that urinary phenanthrene metabolites could be used in a carcinogen metabolite phenotyping approach to identify individuals who may be susceptible to cancer induction by polycyclic aromatic hydrocarbons (PAH). In support of this proposal, we have developed methods for quantitation of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and phenanthrols (HOPhe) in human urine. PheT is the end product of the diol epoxide metabolic activation pathway of PAH, whereas HOPhe are considered as detoxification products. In this study, we investigated the longitudinal consistency of these metabolites over time in smokers and nonsmokers and compared their levels. Twelve smokers and 10 nonsmokers provided urine samples daily for 7 days, then weekly for 6 weeks. Levels of PheT, HOPhe, and PheT/HOPhe ratios were relatively constant in most individuals, with mean coefficients of variation ranging from 29.3% to 45.7%. There were no significant changes over time in levels of the metabolites or in ratios. These results indicate that a single urine sample should be sufficient when comparing phenanthrene metabolites in different groups. PheT/HOPhe ratios were significantly higher in smokers than in nonsmokers, showing that smoking induces the diol epoxide metabolic activation pathway of phenanthrene. This finding is consistent with previous studies indicating that inducibility of PAH metabolism contributes to cancer risk in smokers.

Evaluation of the clinical, immunologic, and biochemical effects of nitroso sulfamethoxazole administration to dogs: a pilot study.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated in humans with hypersensitivity reactions, to include fever, skin eruptions, hepatotoxicity, and blood dyscrasias. These reactions also occur in dogs, the only non-human species known to develop a similar spectrum of sulfonamide hypersensitivity. Sulfonamide hypersensitivity is not well understood, but has been hypothesized to be due to the generation of the reactive oxidative metabolite, nitroso sulfamethoxazole (SMX-NO). SMX-NO, unlike the parent sulfonamide, is cytotoxic in vitro, haptenizes tissue proteins, and is immunogenic in rodents. The purpose of this pilot study was to determine whether SMX-NO, when administered to dogs, would lead to drug-tissue adducts, anti-drug antibodies, antioxidant depletion, or clinical evidence of drug hypersensitivity. Four dogs were randomized to one of four treatments: SMX-NO 1 mg/kg; SMX-NO 3 mg/kg; SMX-NO 10 mg/kg; or vehicle control. Dosing was by the intraperitoneal route, once daily for four consecutive days per week, for 2 weeks total, followed by a third week of observation. Following this, all dogs were challenged with trimethoprim-sulfamethoxazole, 25 mg/kg for 12 h for 2 weeks. No dog developed clinical or biochemical evidence of drug hypersensitivity. Plasma cysteine and leukocyte reduced glutathione were not depleted during dosing; however, ascorbate was significantly depleted by week 2 following SMX-NO at 10 mg/kg. Anti-SMX antibodies (IgG or IgM by ELISA) were not detected in any dogs at any time points. SMX-hemoglobin adducts were detected in the spleen in SMX-NO dosed dogs; however, these adducts were not accompanied by an immunologic or systemic response. The results of this pilot study indicate that SMX-NO dosing in dogs, using a dosing protocol shown to be immunogenic in other species, produces modest ascorbate depletion and hemoglobin adduct formation, but is insufficient to produce an immunologic response or a clinical syndrome of sulfonamide hypersensitivity in this susceptible species.

Plasma ascorbate deficiency is associated with impaired reduction of sulfamethoxazole-nitroso in HIV infection.

OBJECTIVE: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions. METHODS: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro. RESULTS: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%). CONCLUSIONS: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.