Sulfamethoxazole removal and fuel-feedstock biomass production from wastewater in a phyto-Fenton process using duckweed culture.

The phyto-Fenton process, which generates hydroxyl radicals through Fenton and Fenton-like reactions using plant-derived hydrogen peroxide (H2O2) and ferrous iron (Fe (II)) can degrade organic pollutants. Duckweed, an aquatic plant, is promising for a co-beneficial phytoremediation process that combines wastewater treatment and biomass production for biofuel feedstock. However, the phyto-Fenton process using duckweed has not been extensively studied. Because sulfamethoxazole (SMX), a major antibiotic, is distributed widely and is an emerging contaminant, its effective removal from contaminated water is necessary. The present study investigated the possibility of the simultaneous efficient removal of SMX from polluted water and biomass production for fuel feedstock by the phyto-Fenton process using duckweed. This is the first attempt to demonstrate the co-benefits of SMX removal and biomass production using duckweed. Intracellular H2O2 was produced using four duckweeds, Lemna aequinoctialis, L. minor, Landolina punctata, and Spirodela polyrhiza, in the range of 16.7-24.6 µ mol g-1 fresh weight, and extracellular H2O2 was released into the water phase. Consequently, duckweed could be used as an H2O2 supply source for the phyto-Fenton process. Specifically, 0.5 g fresh duckweed almost completely eliminated 1 mg L-1 SMX after 5 d in 50 mL sterile modified Hoagland solution containing 10 mM Fe (II). Fe (II)-dependent elimination of SMX indicated the occurrence of phyto-Fenton reaction. The phyto-Fenton process using duckweed effectively removed SMX. S. polyrhiza duckweed similarly removed 1 mg L-1 SMX even in sewage effluent containing other organic contaminants. During this treatment, duckweed biomass was generated at 7.95 g dry weight m-2 d-1, which was converted into methane at 353 normal liters CH4 kg-1 volatile solids by anaerobic digestion. For the first time, this study clearly demonstrates the potential for simultaneous SMX removal and biomass production from SMX-contaminated wastewater using duckweed.

Reduced graphene oxide promotes the biodegradation of sulfamethoxazole by white-rot fungus Phanerochaete chrysosporium under cadmium stress.

The biodegradation of antibiotics in aquatic environment is consistently impeded by the widespread presence of heavy metals, necessitating urgent measures to mitigate or eliminate this environmental stress. This work investigated the degradation of sulfamethoxazole (SMX) by the white-rot fungus Phanerochaete chrysosporium (WRF) under heavy metal cadmium ion (Cd2+) stress, with a focus on the protective effects of reduced graphene oxide (RGO). The pseudo-first-order rate constant and removal efficiency of 5 mg/L SMX in 48 h by WRF decrease from 0.208 h-1 and 55.6% to 0.08 h-1 and 28.6% at 16 mg/L of Cd2+, while these values recover to 0.297 h-1 and 72.8% by supplementing RGO. The results demonstrate that RGO, possessing excellent biocompatibility, effectively safeguard the mycelial structure of WRF against Cd2+ stress and provide protection against oxidative damage to WRF. Simultaneously, the production of manganese peroxidase (MnP) by WRF decreases to 38.285 U/L in the presence of 24 mg/L Cd2+, whereas it recovers to 328.51 U/L upon the supplement of RGO. RGO can induce oxidative stress in WRF, thereby stimulating the secretion of laccase (Lac) and MnP to enhance the SMX degradation. The mechanism discovered in this study provides a new strategy to mitigate heavy metal stress encountered by WRF during antibiotic degradation.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Assessment of sulfamethoxazole toxicity to marine mussels (Mytilus galloprovincialis): Combine p38-MAPK signaling pathway modulation with histopathological alterations.

Sulfamethoxazole (SMX), is a ubiquitous antibiotic in the aquatic environment and received concerns on its health hazards, especially its sub-lethal effects on non-target organisms which were remained largely unknown. In the present study, in order to investigate SMX induced tissue damages and reveal underlying mechanisms, marine mussels, Mytilus galloprovincialis were challenged to SMX series (0.5, 50 and 500 µg/L) for six-days followed by six-day-recovery. Comprehensive histopathological alteration (including qualitative, semi-quantitative and quantitative indices), together with transcriptional and (post-) translational responses of key factors (p38, NFκB and p53) in the p38-MAPK signaling pathway were analyzed in gills and digestive glands. Tissue-specific responses were clearly investigated with gills showing more prompt responses and digestive glands showing higher tolerance to SMX. The histopathology showed that SMX triggered inflammatory damages in both tissues and quantitative analysis revealed more significant responses, suggesting its potential as a valuable health indicator. SMX activated expressions of p38, NFκB and p53 at transcriptional and (post-) translational levels, especially after exposed to low level SMX, evidenced by p38 coupled with NFκB/p53 regulation on immunity defense in mussels. Less induction of targeted molecules under severe SMX exposure indicated such signaling transduction may not be efficient enough and can result in inflammatory damages. Taken together, this study expanded the understanding of aquatic SMX induced health risk in marine mussels and the underlying regulation mechanism through p38 signaling transduction.

Hepatic and blood alterations in Lithobates catesbeianus tadpoles exposed to sulfamethoxazole and oxytetracycline.

In this study the effects of environmentally realistic concentrations of the antibiotics sulfamethoxazole (SMX) and oxytetracyclyne (OTC) on Lithobates catesbeianus tadpoles were evaluated, through the analyzes of the frequencies of micronucleus and nuclear abnormalities in erythrocytes, alterations in leucocytes, liver histopathology, and changes in hepatic esterase activities and oxidative stress biomarkers. The animals were exposed for 16 days at concentrations of 0 (control), 20, 90 and 460 ng L-1. No significant difference was found in the frequencies of micronucleus and nuclear abnormalities. The two highest concentrations of SMX and all concentrations of OTC caused a significant increase in the number of lymphocytes. A significant decrease in the number of neutrophils compared to the control group was observed for all concentrations tested of both antibiotics. Also, decrease in the activity of glutathione S-transferase and high histopathological severity scores, indicating liver damage, were found in tadpoles exposed to the two highest concentrations of SMX and all concentrations of OTC. The main changes in the liver histopathology were the presence of inflammatory infiltrate, melanomacrophages, vascular congestion, blood cells and eosinophils. Esterase activities were unchanged. Indeed, the two highest concentrations of OTC caused a reduction in the activities of superoxide dismutase and glucose 6-phosphate dehydrogenase, while the highest concentration inhibited the activity of glutathione peroxidase and increased protein carbonyl levels. These results evidences that environmentally realistic concentrations of SMX and OTC in aquatic environments are capable to significantly disrupt tadpoles' physiology, possibly affecting negatively their survival rate in natural environments.

Bio-Pd(0) diverting electron from CoQ-long chain to FDH/Hase-short chain during sulfamethoxazole degradation.

Microbial electron output capacity is critical for organic contaminants biodegradation. Herein, original C. freundii JH could oxidate formate in anaerobic respiration, but lack the ability to degrade sulfamethoxazole (SMX). While the incorporation of Pd(0) could effectively improve the electron output via improving the combination between flavins and c-type cytochromes (c-Cyts), increasing the activities of key enzymes (formate dehydrogenase, hydrogenase, F0F1-ATPases), etc. More importantly, the presence of Pd(0) caused the NADH dehydrogenase (complex I) nearly in idle, and triggered the decrease of NADH/NAD+ ratio and increase of H+-efflux transmembrane gradient, eventually resulting in the electrons diverting from CoQ-involved long respiratory chain (decreasing from 91.67% to 36.25%) to FDH/Hases-based hydrogen-producing short chain (increasing from 22.44% to 84.88%), which further intensified the electron output. Above changes effectively launched and guaranteed the high-level SMX degradation by palladized C. freundii JH, alleviating the ecotoxicity of SMX in aquatic and terrestrial environments. These conclusions provided the new view to regulate the microbial electron output behaviors.

Toxic effects and molecular mechanisms of sulfamethoxazole on Scenedesmus obliquus.

The antibiotic sulfamethoxazole (SMX) is a pollutant that is widely distributed in the global water environment.This substance has toxic effects on various aquatic organisms. Previous studies on SMX have focused on its acute toxicity towards algae and the changes induced at biological and cellular levels, rather than its biotoxicity and mechanisms at the molecular level. In this study, we investigated the effects of SMX on Scenedesmus obliquus as the model organism by performing transmission electron microscopy and transcriptome sequencing analyses. Exposure to SMX promoted gene expression, resulting in changes to algal cell ultrastructure. The cell walls became blurred, the chloroplast structure was seriously damaged, and the number and volume of mitochondria per cell increased. These changes were related to the inhibition of cell growth, decrease in chlorophyll content, increase in cell membrane permeability, and increased production of reactive oxygen species, which led to increased amounts of the lipid peroxidation product malondialdehyde, and higher activities of antioxidant enzymes. Our results suggest that SMX affects gene expression by influencing non-coding RNA metabolic processes, leading to changes in nuclear structures. Abnormally expressed long non-coding RNAs extensively regulate downstream gene expression through various mechanisms, such as chromatin recombination, thereby promoting tumor occurrence, invasion, and metastasis. This abnormal expression may be an important mechanism underlying the carcinogenic effects of SMX.

Oxidative stress is involved in the activation of NF-κB signal pathway and immune inflammatory response in grass carp gill induced by cypermethrin and/or sulfamethoxazole.

At present, the concentration of environmental pollutants, such as pesticides and antibiotics exposed in environment, especially in aquatic environment is increasing. Research on environmental pollutants has exploded in the last few years. However, studies on the combined effects of pesticides and antibiotics on fish are rare, especially the toxic damage to gill tissue is vague. In this paper, cypermethrin (CMN) and sulfamethoxazole (SMZ) were analyzed and found that there was a strong correlation between the pathways affected by the first 30 genes regulated by CMN and SMZ, respectively. Therefore, the toxic effects of CMN (0.651 µg L-1) and/or SMZ (0.3 µg L-1) on grass carp gill were studied in this paper. Histopathology, quantitative real-time PCR, and other methods were used to detect the tissue morphology, oxidative stress level, inflammation, and apoptosis-related indicators of the fish gills after exposure of 42 days. It was found that compared with the single exposure (CMN/SMZ) group, the combined exposure (MIX) group had a more pronounced oxidative stress index imbalance. At the same time, nuclear factor-κB (NF-κB) signal pathway was activated and immuno-inflammatory reaction appeared in MIX group. The expression of tumor necrosis factor (TNF-α) in the rising range is 2.94 times that of the C group, while the expression of interleukin 8 (IL-8) is as high as 32.67 times. This study reveals the harm of CMN and SMZ to fish, and provides a reference and basis for the rational use of pesticides and antibiotics.

Inflammatory response and oxidative stress attenuated by sulfiredoxin­1 in neuron­like cells depends on nuclear factor erythroid­2­related factor 2.

Sulfiredoxin­1 (SRX1) is a conserved endogenous antioxidative protein, which is involved in the response to cellular damage caused by oxidative stress. Oxidative stress and inflammation are the primary pathological changes in spinal cord injuries (SCI). The aim of present study was to explore the roles of SRX1 in SCI. Using reverse transcription­quantitative PCR and western blotting, the present study discovered that the expression levels of SRX1 were downregulated in the spinal cord tissues of SCI model rats. Massive irregular cavities and decreased Nissl bodies were observed in the model group compared with the sham group. Thus, to determine the underlying mechanisms, neuron­like PC12 cells were cultured in vitro. Western blotting analysis indicated that SRX1 expression levels were downregulated following the exposure of cells to lipopolysaccharide (LPS). Following the transfection with the SRX1 overexpression plasmid and stimulation with LPS, the results of the Cell Counting Kit­8 assay indicated that the cell viability was increased compared with LPS stimulation alone. Furthermore, the expression levels of proinflammatory cytokines secreted by LPS­treated PC12 cells were downregulated following SRX1 overexpression. Increased malondialdehyde content, decreased superoxide dismutase activity and reactive oxygen species production were also identified in PC12 cells treated with LPS using commercial detection kits, whereas the overexpression of SRX1 partially reversed the effects caused by LPS stimulation. The aforementioned results were further verified by determining the expression levels of antioxidative proteins using western blotting analysis. In addition, nuclear factor erythroid­2­related factor 2 (NRF2), a transcription factor known to regulate SRX1, was indicated to participate in the protective effect of SRX1 against oxidative stress. Inhibition of NRF2 further downregulated the expression levels of SRX1, NAD(P)H dehydrogenase quinone 1 and heme oxygenase­1 in the presence of LPS, while activation of NRF2 reversed the effects of LPS on the expression levels of these proteins. In conclusion, the results of the present study indicated that the anti­inflammatory and antioxidative effects of SRX1 may depend on NRF2, providing evidence that SRX1 may serve as a novel molecular target to exert a neuroprotective effect in SCI.

Evaluation of bezafibrate, gemfibrozil, indomethacin, sulfamethoxazole, and diclofenac removal by ligninolytic enzymes.

The laccase (Lac), manganese peroxidases (MnP), and lignin peroxidase enzymes produced by basidiomycete have been studied due to their potential in bioremediation, therefore, in this study, degradation of diclofenac (DCF), sulfamethoxazole (SMX), indomethacin (IND), gemfibrozil (GFB), and bezafibrate (BZF) by enzymes produced by Trametes maxima, Pleurotus sp., and Pycnosporus sanguineus grown in culture was evaluated. The degradation of drugs can mainly be attributed to MnP because a correlation between the activity of this enzyme and the degree of removal was found. The specific activity of Lac did not show correlation with drug removal, while lignin peroxidase was not expressed. Trametes maxima showed the highest specific activity of MnP (387.6 ± 67.4 U/mg) and efficiency removal 90.2% of DCF, 72.62% of SMX, 60.76% of IND, 43.39% of GFB, and 32.59% of BZF) followed by Pleurotus sp. with specific activity of MnP of 55.9 ± 8.5 U/mg and 89.47% of DCF, 47.61% of GFB and 73% of IND were removed, P. sanguineus had the lowest specific activity of 18 ± 1.3 U/mg and was able to remove only 42% of SMX and 10.59% of IND. In order to prove that MnP remove drugs instead of Lac, the pure Lac was tested and only degraded DCF.

Anaerobic biodegradation of pharmaceutical compounds: New insights into the pharmaceutical-degrading bacteria.

Antibiotics and hormones are among the most concerning trace contaminants in the environment. Therefore, the present work aimed to identify anaerobic microorganisms with the ability to remove pharmaceutical products (PhPs) belonging to these two classes (ciprofloxacin, 17ß-estradiol and sulfamethoxazole) under different anaerobic conditions, and to elucidate the bio-removal mechanisms involved. Ciprofloxacin was efficiently biodegraded under both nitrate- and sulfate-reducing conditions reaching a PhP removal superior to 80%, whereas 17ß-estradiol was only biodegraded under nitrate-reducing conditions reaching a removal of 84%. No biodegradation of sulfamethoxazole was observed. In nitrate-reducing conditions the ciprofloxacin-degrading community was composed of Comamonas, Arcobacter, Dysgonomonas, Macellibacteroides and Actinomyces, genera while Comamonas and Castellaniella were the main bacteria present in the 17ß-estradiol-degrading community. In sulfate-reducing conditions the community was mainly composed by bacteria affiliated to Desulfovibrio, Enterococcus and Peptostreeptococcus. Interestingly, the PhP under study were biodegraded even in the absence of additional carbon source, with 85% of ciprofloxacin removed under sulfate-reducing conditions and 62% and 83% of ciprofloxacin and estradiol removed, respectively, under nitrate-reducing conditions. This work provides new insights into anaerobic bioremediation of PhP and novel PhP-degrading bacteria.

Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD+) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP+ are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP+ ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.

Rapid degradation of sulphamethoxazole and the further transformation of 3-amino-5-methylisoxazole in a microbial fuel cell.

Sulphamethoxazole (SMX) is extensively used in humans and livestock, but its appearance in natural water raises environmental concerns. This study demonstrated that SMX and its degradation product, 3-amino-5-methylisoxazole (3A5MI), could be effectively degraded in microbial fuel cell (MFC) reactors. Approximately 85% of 20 ppm SMX was degraded within 12 h, and this was a more rapid biodegradation rate than has been previously shown in the literature. In addition, 3A5MI, a toxic chemical that forms in the SMX degradation process, can be further mineralized. The degradation products of SMX were detected by mass spectrometry, and three speculated by-products were confirmed with chemical standards. It was observed that nitrogen atoms of SMX were progressively eliminated during the degradation process, which may relate with the degradation of SMX and 3A5MI. An antibacterial activity test showed that the biotoxicity of SMX towards Shewanella oneidensis MR-1 and Escherichia coli DH5α was greatly reduced after MFC treatment. Moreover, the ATP level of the MFC microbe was nearly threefold higher than that in open-circuit controls, which may be related to the rapid degradation of SMX in MFCs. This study can facilitate further investigations about the biodegradation of SMX.

Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVß20-1, altering pHLA recognition.

T cell receptors (TCR) containing Vß20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vß20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2ß domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vß20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable ß or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2ß loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2ß loop amino acid positions. These residues, TYRß57, ASPß64, and LYSß65 initially hold hydrogen bonds from the CDR2ß to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2ß of Vß20-1 acts as a ligand controlled switch affecting overall TCR binding affinity.

Polymorphism in glutamate cysteine ligase catalytic subunit (GCLC) is associated with sulfamethoxazole-induced hypersensitivity in HIV/AIDS patients.

BACKGROUND: Sulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity. RESULTS: Seventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 - 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 - 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05). CONCLUSIONS: rs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions.

Microbially mediated abiotic transformation of the antimicrobial agent sulfamethoxazole under iron-reducing soil conditions.

Large quantities of antimicrobial agents used in livestock production are released to soils by land application of manure, but only limited information is available on mechanisms that contribute to antimicrobial fate in soils under variable biogeochemical conditions. Dissipation of the sulfonamide antimicrobial sulfamethoxazole was examined in soil microcosms incubated under different terminal electron-accepting conditions (aerobic, nitrate-reducing, Fe(III)-reducing, and sulfate-reducing). Somewhat unexpectedly, sulfamethoxazole dissipation was fastest under Fe(III)-reducing conditions, with concentrations decreasing by >95% within 1 day. The rapid transformation was attributed to abiotic reactions between sulfamethoxazole and Fe(II) generated by microbial reduction of Fe(III) soil minerals. Separate experiments demonstrated that sulfamethoxazole was abiotically transformed in Fe(II)-amended aqueous suspensions of goethite (α-FeOOH((s))), and observed rate constants varied with the extent of Fe(II) sorption to goethite. Sulfamethoxazole transformation is initiated by a 1-electron reductive cleavage of the N-O bond in the isoxazole ring substituent, and observed products are consistent with Fe(II)-mediated reduction and isomerization processes. These findings reveal potentially important, but previously unrecognized, pathways that may contribute to the fate of sulfamethoxazole and related chemicals in reducing soil environments.

Enhanced antigenicity leads to altered immunogenicity in sulfamethoxazole-hypersensitive patients with cystic fibrosis.

BACKGROUND: Exposure of patients with cystic fibrosis to sulfonamides is associated with a high incidence of hypersensitivity reactions. OBJECTIVE: To compare mechanisms of antigen presentation and characterize the phenotype and function of T cells from sulfamethoxazole-hypersensitive patients with and without cystic fibrosis. METHODS: T cells were cloned from 6 patients and characterized in terms of phenotype and function. Antigen specificity and mechanisms of antigen presentation to specific clones were then explored. Antigen-presenting cell metabolism of sulfamethoxazole was quantified by ELISA. The involvement of metabolism in antigen presentation was evaluated by using enzyme inhibitors. RESULTS: Enzyme inhibitable sulfamethoxazole-derived protein adducts were detected in antigen-presenting cells from patients with and without cystic fibrosis. A significantly higher quantity of adducts were detected with cells from patients with cystic fibrosis. Over 500 CD4(+) or CD8(+) T-cell clones were generated and shown to proliferate and kill target cells. Three patterns of MHC-restricted reactivity (sulfamethoxazole-responsive, sulfamethoxazole metabolite-responsive, and cross-reactive) were observed with clones from patients without cystic fibrosis. From patients with cystic fibrosis, sulfamethoxazole metabolite-responsive and cross-reactive, but not sulfamethoxazole-responsive, clones were observed. The response of the cross-reactive clones to sulfamethoxazole was dependent on adduct formation and was blocked by glutathione and enzyme inhibitors. Antigen-stimulated clones from patients with cystic fibrosis secreted higher levels of IFN-γ, IL-6, and IL-10, but lower levels of IL-17. CONCLUSION: Sulfamethoxazole metabolism and protein adduct formation is critical for the stimulation of T cells from patients with cystic fibrosis. T cells from patients with cystic fibrosis secrete high levels of IFN-γ, IL-6, and IL-10.

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis.

AIMS: Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b(5) (b5) and cytochrome b(5) reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens. METHODS: Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly. RESULTS: There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µm vs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r= 0.39, P= 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg(-1) min(-1)) than controls (18.9 µmol mg(-1) min(-1), P= 0.008). After 3-4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash. CONCLUSIONS: Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.

Drug metabolite-specific lymphocyte responses in sulfamethoxazole allergic patients with cystic fibrosis.

Sulfamethoxazole (SMX) is an important antibiotic in the management of patients with cystic fibrosis, but allergic reactions may develop thus restricting therapy. The aim of this study was to utilize drug (metabolite) antigens to diagnose SMX-mediated allergic reactions in patients with cystic fibrosis. Lymphocytes from 2/12 allergic patients were stimulated to proliferate strongly with the SMX metabolite nitroso SMX (SMX-NO). In contrast, responses to SMX were weak. The introduction of an antigen-driven T-cell enrichment step prior to the analysis of proliferation increased the sensitivity of the assay. SMX-NO responses were detected with lymphocytes from all patients with cutaneous signs.

Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.