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Sulfonamides, recognized as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, are crucial in treating diverse diseases, including epilepsy, glaucoma, bacterial infections, and various pathological processes, e.g., high blood pressure, rheumatoid arthritis, ulcerative colitis, pain, and inflammation. Additionally, therapeutically, 1,3-diaryl-substituted triazenes and sulphamethazines (SM) are integral components in various drug structures, and the synthesis of novel compounds within these two categories holds substantial significance. Herein, ten 1,3-diaryltriazene-substituted sulphamethazine derivatives SM(1-10), which were created by reacting the diazonium salt of sulphamethazine with substituted aromatic amines, were synthesized and the physiologically and pharmacologically relevant human (h) isoforms hCA I and II, cytosolic isozymes, were included in the study. The synthesized compounds showed excellent inhibition versus hCAs; the 4-butoxy (SM7, K I of 5.69 ± 0.59 nM) compound exhibited a potent inhibitory effect against the hCA I compared with the reference drug acetazolamide (AAZ, K I of 116.00 ± 8.48 nM). The 4-cyano (SM4, K I of 5.87 ± 0.57 nM) compound displayed higher potency than AAZ (K I of 57.25 ± 4.15 nM) towards hCA II. Meanwhile, among the synthesized molecules, the 3,4-dimethoxy (SM9, K I of 74.98 ± 10.49 nM, S I of 9.94) compound (over hCA I) displayed a noticeable selectivity for hCA isoform II. The target compounds in the molecular docking investigation were determined to take part in various hydrophilic and hydrophobic interactions with nearby amino acids and fit nicely into the active sites of the hCAs. This research has yielded compounds displaying varying affinity toward hCA isoenzymes, ultimately serving as potent and selective hCA inhibitors. Given its substantial biological inhibitory potency, this particular derivative series is determined to hold the potential to serve as a promising lead compound against these hCAs., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aim: The purpose of this study is to design and synthesize a new series of sulfamethazine derivatives as potent neuraminidase inhibitors. Materials & methods: A sulfamethazine lead compound, ZINC670537 , was first identified by structure-based virtual screening technique, then some novel inhibitors X1-X10 based on ZINC670537 were designed and synthesized. Results: Compound X3 exerts the most good potency in inhibiting the wild-type H5N1 NA (IC 50  = 6.74 μM) and the H274Y mutant NA (IC 50  = 21.09 μM). 150-cavity occupation is very important in determining activities of these inhibitors. The sulfamethazine moiety also plays an important role. Conclusion: Compound X3 maybe regard as a good anti-influenza candidate to preform further study.

Antibiotic contamination from animal production and wastewater treatment process will release antibiotic resistant genes to the environment and potentially threaten human health. Meanwhile, the residual antibiotic in manure could have inactive impacts on anaerobic digestion (AD). This study explores the effect of sulfamethazine on manure AD mediated by biochar. The results show that biochar weakens the adverse effects of sulfamethazine on AD by adsorption sulfamethazine during the initial stage (0-3 days) of AD and promoting the growth of hydrolytic bacteria (especially Firmicutes and Bacteroidetes) and methanogens (especially Methanothrix and Methanosarcina). Besides, the presence of biochar improves the biogas production capacity of AD and promotes microbial diversity and community richness. Thus, the addition of biochar greatly reduces sulfamethazine and is testified to be a desirable strategy to mitigate the inhibition of sulfamethazine on AD., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microplastics in the environment can be colonized by microbes capable of forming biofilms, which may act as reactive coatings to affect the bioaccessibility of pollutants in organisms. This study investigated the dynamic evolution of biofilm colonization on microplastics and its impacts and mechanisms on the bioaccessibility of microplastic-associated sulfamethazine (SMT) via microcosm incubation in surface water and sediment. After 60 days of incubation, the microbial communities formed in microplastics were distinct and more diverse than those untethered in surroundings, and photoaging treatment decreased the affinity of biofilms on microplastics due to decreased hydrophobicity. Biofilm formation further enhanced the desorption and bioaccessibility of microplastic-sorbed SMT in organisms. In vitro experiments indicated that the critical effects were mainly related to the stronger interaction of gastrointestinal components (i.e., pepsin, bovine serum albumin (BSA), and NaT) with biofilm components (e.g., extracellular polymer substances) than with the pure surface of microplastics, which competed for binding sites in microplastics for SMT more significantly. Photoaging decreased the enhancing effects of biofilms due to their lower accumulation in aged microplastics. This study is the first attempt to reveal the role of biofilms in the bioaccessibility of microplastics with associated antibiotics and provide insights into the combined risk of microplastics in the environment.

Heavy metals and antibiotics residues in agricultural soils are attracting more and more attention. A laboratory study was conducted to evaluate the single and combined effects of sulfadimidine (SM2) (0.05, 0.20, 0.80 mmol/kg) and copper (Cu) (1.60 mmol/kg) on soil microbial biomasses and ammoxidation microorganisms abundances after 7, 14, 21 and 28 days. The results demonstrated that the single and combined contaminations had a significant and persistent inhibitory effect on soil bacteria, fungi and actinomycetes populations and amoA gene copies of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) (Except SM2 0.05 and 0.20 mmol/kg on 7 and 14 d and SM2 0.05 mmol/kg on 21 d led to a stimulatory effect on fungi and AOA-amoA gene, respectively). With higher dosage and longer exposure time, the toxic effect of single and combined contaminants on soil bacteria, fungi and actinomycetes as well as on the amoA gene of AOA and AOB was greatly reinforced. Combined contaminants produced more toxicity than the chemicals were used alone. Overall, the interaction effects of SM2 and Cu on bacteria (on 14, 21 and 28 d), fungi and AOA-amoA were mainly synergism, in contrast, on actinomycetes (on 14, 21 and 28 d) and AOB-amoA were mainly antagonism. The order of the toxic effects of the single Cu and combined contaminants on microbial activity was: bacteria > actinomycetes > fungi. Furthermore, AOB-amoA was more sensitive to both contaminants toxicity than AOA-amoA, while AOA-amoA gene copies were greater than AOB-amoA gene copies about one order of magnitude., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Antibiotics enter into aquatic pond sediments by wastewater and could make detrimental effects on microbial communities. In this study, we examined the effects of sulfadimidine on nitrogen removal when added to experimental pond sediments. We found that sulfadimidine increased the number of sulfadimidine resistant bacteria and significantly increased the abundance of sul2 at the end of the incubation time (ANOVA test at Tukey HSD, P < 0.05). In addition, sulfadimidine decreased the N 2 O reduction rate as well as the amount of nitrate reduction. Pearson correlation analysis revealed that the N 2 O reduction rate was significantly and negatively correlated with narG (r = -0.679, P < 0.05). In contrast, we found a significant positive correlation between the amount of nitrate reduction and the abundance of narG (r = 0.609, P < 0.05) and nirK (r = 0.611, P < 0.05). High-throughput sequencing demonstrated that Actinobacteria, Euryarchaeota, Gemmatimonadetes, Nitrospirae, Burkholderiaceae (a family of Proteobacteria), and Thermoanaerobaculaceae (a family of Firmicutes) decreased with sulfadimidine exposure. In sediments, Actinobacteria, Bacteroidetes, Cyanobacteria, Epsilonbacteraeota, Euryarchaeota, Firmicutes, Gemmatimonadetes, and Spirochaetesat may play key roles in nitrogen transformation. Overall, the study exhibited a net effect of antibiotic exposure regarding nitrogen removal in an aquatic microcosm environment through a combination of biochemical pathways and molecular pathways, and draws attention to controlling antibiotic pollution in aquatic ecosystems., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study evaluated how tylosin (TYL), ciprofloxacin (CIP), and sulfadimidine (SM 2 ) affected biogas and CH 4 production during anaerobic digestion (AD) via their effects on the key genes related to methane production and the methanogenic community. The results showed that TYL, CIP, and SM 2 reduced the production of methane during AD by 7.5%, 21.9%, and 16.0%, respectively. After AD for five days, CIP strongly inhibited the mcrA gene, where its abundance was 49% less than that in the control. TYL and SM 2 decreased the abundances of Spirochaeta and Fibrobacteres during AD. High-throughput sequencing identified 10 methanogen genera, where Methanocorpusculum, Methanobrevibacter, and Methanosarcina accounted for 99.1% of the total archaeal reads. TYL and SM 2 increased the efficiency of the acetoclastic methanogen pathway (Methanosarcina) by 29.04% and 52.79%, respectively. Redundancy analysis showed that Spirochaeta, Fibrobacteres, and Methanosarcina had positive correlations with CH 4 and mcrA. We found that 30 mg kg -1 CIP had a strong inhibitory effect on methane production by influencing the abundances of Methanobrevibacter and Methanosarcina during AD., (Copyright © 2018. Published by Elsevier Ltd.)

The study analyzed the correlation between the antibiotic-induced feeding depression and body size reduction in rotifer, Brachionus calyciflorus, involving exposure, post-exposure and re-exposure periods. The filtration and ingestion rates of the rotifers were inhibited in these three exposure periods at any given concentration of the antibiotic sulfamethazine (SMZ). As food for rotifer, the cell size of the green algae was unchanged, which indicated that it could not drive feeding depression. Secondly, several corresponding physiological responses were considered. Reactive oxygen species (ROS) levels increased in the post-exposure and the re-exposure; acetylcholinesterase (AChE) activity was significantly decreased in the exposure and the re-exposure, whereas it was induced in the post-exposure. The activities of amylase and lipase were always inhibited in these three exposure periods. Additionally, significant decreases in lorica length, width and biovolume of rotifers occurred after the feeding depression. Statistical analysis indicated a positive correlation between the activity of the digestive enzyme and the body size. Our results demonstrated that SMZ could influence the neurotransmission, inhibit the activity of the digestive enzyme, and finally result in body size reduction. These results provided an integrated perspective on assessing the toxicity effects of antibiotic in non-lethal dosage on the feeding behavior of non-target aquatic organisms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ideal drugs to cure cancer leave normal cells unharmed while selectively turning tumor cells unviable. Several copper complexes have been able to selectively slow down tumor proliferation. We hypothesized that Cu(smz) 2 (bipy)·H 2 O (1)-a copper-complex that has two ligands capable of interacting with DNA-would outperform Cu(smz) 2 (OH 2 )·2H 2 O (2), and also that supporting 1 on mesoporous silica spheres would decrease even further tumor cell viability in vitro. After exposing osteosarcoma cells (MG-63) and normal phenotype cells of bone origin (MC3T3-E1) to either complex, we studied their toxic effect and mechanisms of action. We determined cell viability (MTT assay) and quantified formation of reactive oxygen species (oxidation of DHR-123 to rhodamine). Moreover, we assessed genotoxicity from (i) formation of micronucleus (MN assay) and (ii) damage of DNA (Comet assay). After the exposure of 1 supported on silica spheres, we tested cell viability. Our results confirm our hypotheses: inhibition of tumor cells follows: supported 1 > dissolved 1 > 2. Future work that enhances the load of the complex exclusively in mesopores may improve the ability of 1 to further inhibit tumor cell viability.

Sulfamethazine (SMZ) (1) is an antibacterial sulfa drug which suppresses the synthesis of dihydrofolic acid. It is used for the treatment of infections in livestock; such as gastrointestinal, and respiratory tract infections. During the current study, synthesis, characterization, and evaluation of immunomodulatory activities of derivatives of sulfamethazine (SMZ) (3-39) was carried out. These derivatives were synthesized by the reaction of sulfamethazine with a range of acid chlorides. All the compounds were characterized by using modern spectroscopic techniques, such as 1H-, and 13C-NMR, EI-MS, and HRFAB-MS. Compounds 3-10, 14, and 15 were identified as new compounds. Immunomodulatory effect of compounds 3-39 on different parameters of innate immune response was evaluated, including the production of Reactive Oxygen Species (ROS) from human whole blood and isolated polymorphonuclear neutrophils (PMNs), nitric oxide (NO), and pro-inflammatory cytokine TNF-α. All the new compounds, except 14 and 15, showed a significant anti-inflammatory activity. Compounds 3-39 were also evaluated for their anti-bacterial activity and cytotoxicity (3T3 mouse fibroblast cell lines). All the compounds were found to be non-cytotoxic against normal cell lines., Competing Interests: The authors have declared that no competing interests exist.

Sulfadimidine (SM2) is commonly used in the swine industry and enters the environment via faeces. In recent years, advances in the ecotoxicology of SM2 have become a popular research interest with two common research methods including swine manure collection from swine fed with a diet containing SM2 and directly adding SM2. The purpose of this experiment was to compare SM2 degradation behaviour in pig manure with two different SM2 addition methods. The results showed that the degradation half-lives of SM2 in manure from SM2-fed swine treatment were 33.2 and 32.0 days at the initial addition level of SM2 at 32.1 and 64.3 mg/kg, respectively. This was significantly longer than that in manure directly adding SM2 treatment with the half-lives of 21.4 and 14.8 days. The metabolite of SM2 N 4 -acetyl-sulfamethazine occurred in manure from SM2-fed swine treatment but was not detected in directly adding SM2 treatment. The pH in manure from SM2-fed swine treatment was significantly lower than that in directly adding SM2 treatment, but the values of organic carbon, total nitrogen, and electrical conductivity in manure from SM2-fed swine treatment were significantly higher than those in manure directly adding SM2 treatment. Meanwhile, although the copy number of bacteria had no significant difference between two treatments, there was a significant difference in bacteria diversity. Results of the present study demonstrated that the presence of the metabolites, chemical property, and microbial diversity might be the reason for different SM2 degradation behaviours on different addition methods. Thus, the method using manure with SM2 collected from swine could obtain more accurate results for the ecotoxicological study of SM2.

Unlabelled: Transcatheter arterial chemoembolization (TACE) is the most common palliative therapy for unresectable hepatocellular carcinoma (HCC). The conventional TACE technique, which employs the Lipiodol® emulsion, has been widely used for human cancer treatments. However, this delivery system seems to be inconsistent and unstable in maintaining a high concentration of drugs at tumor sites. An alternative approach for TACE is loading drugs into a liquid embolic solution that exists as an injectable solution and can exhibit a sol-to-gel phase transition to form a solidified state once delivered to the tumor site. Here, we develop a novel sulfamethazine-based anionic pH-sensitive block copolymer with potential application as a radiopaque embolic material. The copolymer, named PCL-PEG-SM, and comprised of poly(ε-caprolactone), sulfamethazine, and poly(ethylene glycol), was fabricated by free radical polymerization. An aqueous solution of the developed copolymer underwent a sol-to-gel phase transition upon lowering the environmental pH to create a gel region that covered the physiological condition (pH 7.4, 37°C) and the low pH conditions at tumor sites (pH 6.5-7.0, 37°C). The release of doxorubicin (DOX) from DOX-loaded copolymer hydrogels could be sustained for more than 4weeks in vitro, and the released DOX retained its fully bioactivity via inhibition the proliferation of hepatic cancer cells. The radiopaque embolic formulations that were prepared by mixing copolymer solutions at pH 8.0 with Lipiodol®, a long-lasting X-ray contrast agent, could exhibit the gelation inside the tumor after intratumoral injection or intraarterial administration using a VX2 carcinoma hepatic tumor rabbit model. These results suggest that a novel anionic pH-sensitive copolymer has been developed with a potential application as a liquid radiopaque embolic solution for TACE of HCC., State of Significance: Transcatheter arterial chemoembolization (TACE) has been widely used as a palliative treatment therapy for unresectable hepatocellular carcinoma (HCC). Conventional TACE technique, which usually employs emulsion of DOX-in-Lipiodol®, followed by an embolic agent, has significant limitation of inconsistency and lack of controlled release ability. To address these limitations of conventional TACE material system, we introduced a novel liquid radiopaque embolic material from our pH-sensitive hydrogel. The material has low viscosity that can be injected via a microcatheter, rather biocompatibility, and drug controlled release ability. Importantly, it can form gel in the tumor as well as tumoral vasculature in response to the lowered pH at the tumor site, which proved the potential for the use to treat HCC by TACE therapy., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Purpose: The aim of this work was to evaluate the influence of crystal habit on the dissolution and in vitro antibacterial and anitiprotozoal activity of sulfadimidine:4-aminosalicylic acid cocrystals., Methods: Cocrystals were produced via milling or solvent mediated processes. In vitro dissolution was carried out in the flow-through apparatus, with shadowgraph imaging and mechanistic mathematical models used to observe and simulate particle dissolution. In vitro activity was tested using agar diffusion assays., Results: Cocrystallisation via milling produced small polyhedral crystals with antimicrobial activity significantly higher than sulfadimidine alone, consistent with a fast dissolution rate which was matched only by cocrystals which were milled following solvent evaporation. Cocrystallisation by solvent evaporation (ethanol, acetone) or spray drying produced flattened, plate-like or quasi-spherical cocrystals, respectively, with more hydrophobic surfaces and greater tendency to form aggregates in aqueous media, limiting both the dissolution rate and in vitro activity. Deviation from predicted dissolution profiles was attributable to aggregation behaviour, supported by observations from shadowgraph imaging., Conclusions: Aggregation behaviour during dissolution of cocrystals with different habits affected the dissolution rate, consistent with in vitro activity. Combining mechanistic models with shadowgraph imaging is a valuable approach for dissolution process analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Surface water samples from downstream and estuarine areas of Jiulong River were collected in August 2011 and May 2012 for detecting sulfonamide antibiotic residues and isolating sulfamethazine-resistant bacteria. Sulfamethazine was detected in all samples in May 2012 at an average concentration of 78.3 ng L(-1), which was the highest among the nine sulfonamide antibiotics determined. Sulfamethazine-resistant bacteria (SRB) were screened using antibiotic-containing agar plates. The SRB average abundance in the samples was 3.69 × 10(4) and 2.17 × 10(3) CFUs mL(-1) in August 2011 and May 2012, respectively, and was positively correlated to sulfamethazine concentration in May 2012. The 16S rRNA gene sequencing of all the 121 SRB isolates revealed high diversity. Furthermore, the SRB isolates exhibited multidrug resistance, with 48.7% showing resistance to at least three antibiotics. The abundance and persistence of highly diverse SRB and their multidrug resistance are likely to demonstrate the transferable pressure from coastal environments on public health.

Three primitive photoreceptors [melanopsin (Opn4), neuropsin/opsin5 (Opn5) and vertebrate ancient opsin (VAOpn)] were reported as possible avian deep-brain photoreceptors (DBPs) involved in the perception of photoperiodic information affecting the onset and development of reproduction. The objective of this study was to determine the effect of long-day photostimulation and/or sulfamethazine treatment (SMZ, a compound known to advance light-induced testes development) on gene expression of DBPs and key hypothalamic and pituitary genes involved in avian reproductive function. Two-week old chicks were randomly selected into four experimental groups: short-day control (SC, LD8:16), short-day+SMZ (SS, LD8:16, 0.2% diet SMZ), long-day control (LC, LD16:8), and long-day+SMZ (LS, LD16:8, 0.2% diet SMZ). Birds were sampled on days 3, 7, and 28 after initiation of a long-day photoperiod and/or SMZ dietary treatments. Three brain regions [septal-preoptic, anterior hypothalamic (SepPre/Ant-Hypo) region, mid-hypothalamic (Mid-Hypo) region, posterior-hypothalamic (Post-Hypo) region], and anterior pituitary gland were dissected. Using quantitative real-time RT-PCR, we determined changes of expression levels of genes in distinct brain regions; Opn4 and Opn5 in SepPre/Ant-Hypo and Post-Hypo regions and, VAOpn in the Mid-Hypo region. Long-day treatment resulted in a significantly elevated testes weight on days 7 and 28 compared to controls, and SMZ augmented testes weight in both short- and long-day treatment after day 7 (P<0.05). Long-day photoperiodic treatment on the third day unexpectedly induced a large 8.4-fold increase of VAOpn expression in the Mid-Hypo region, a 15.4-fold increase of Opn4 and a 97.8-fold increase of Opn5 gene expression in the Post-Hypo region compared to SC birds (P<0.01). In contrast, on days 7 and 28, gene expression of the three DBPs was barely detectable. LC group showed a significant increase in GnRH-1 and TRH mRNA in the Mid-Hypo compared to SC on day 3. Pituitary LHβ and FSHβ mRNA were significantly elevated in LC and LS groups compared to SC on days 3 and 7 (P<0.05). On days 3 and 7, TSHβ mRNA level was significantly elevated by long-day treatment compared to the SC groups (P<0.05). Results suggest that long-day photoperiodic activation of DBPs is robust, transient, and temporally related with neuroendocrine genes involved in reproductive function. Additionally, results indicate that two subsets of GnRH-1 neurons exist based upon significantly different gene expression from long-day photostimulation and long-day plus SMZ administration. Taken together, the data indicate that within 3 days of a long-day photoperiod, an eminent activation of all three types of DBPs might be involved in priming the neuroendocrine system to activate reproductive function in birds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Denitrification is an important pathway of nitrogen removal and nitrous oxide (N2O) production in estuarine and coastal ecosystems, and plays a significant role in counteracting aquatic eutrophication induced by excessive nitrogen loads. Estuarine and coastal environments also suffer from increasing antibiotic contamination because of the growing production and usage of antibiotics. In this study, sediment slurry incubation experiments were conducted to determine the influence of sulfamethazine (SMT, a sulphonamide antibiotic) on denitrification and the associated N2O production. Genes important for denitrification and antibiotic resistance were quantified to investigate the microbial physiological mechanisms underlying SMT's effects on denitrification. SMT was observed to significantly inhibit denitrification rates, but increasing concentrations of SMT enhanced N2O release rates. The negative exponential relationships between denitrifying gene abundances and SMT concentrations showed that SMT reduced denitrification rates by restricting the growth of denitrifying bacteria, although the presence of the antibiotic resistance gene was detected during the incubation period. These results imply that the wide occurrence of residual antibiotics in estuarine and coastal ecosystems may influence eutrophication control, greenhouse effects, and atmospheric ozone depletion by inhibiting denitrification and stimulating the release of N2O.

In the present work, a combined experimental and theoretical study of the N-(4,6-Dimethyl-pyrimidin-2-yl)-4-[(2-hydroxy-benzylidene)amino]benzenesulfonamide ligand (H2L) and its mononuclear and magnetically diluted binuclear Cu(II) complexes has been performed using IR, TG/DTA, magnetic, EPR, and conductivity measurements. Calculated g-tensor values showed best agreement with experimental values from EPR when carried out using the MPW1PW91 functional. Coordination of H2L to a Cu(II) center, regardless of the binding site and Cu:L stoichiometry, leads to a significant decrease in the antibacterial activity compared to the free ligand as well as reference drugs in the case of Staphylococcus aureus. Structural-activity relationship suggests that ELUMO, ΔE, dipole moment, polarizability and electrophilicity index were the most significant descriptors for the correlation with the antibacterial activity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR'). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a-5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b-5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds.

The impacts of four common animal husbandry antibiotics (ampicillin, florfenicol, sulfamethazine, and tylosin) on anaerobic digestion (AD) treatment efficiency and the potential for antibiotic degradation during digestion were evaluated. Sulfamethazine and ampicillin exhibited no impact on total biogas production up to 280 and 350 mg/L, respectively, although ampicillin inhibited biogas production rates during early stages of AD. Tylosin reduced biogas production by 10-38% between 130 and 913 mg/L. Florfenicol reduced biogas by ≈ 5%, 40% and 75% at 6.4, 36 and 210 mg/L, respectively. These antibiotic concentrations are higher than commonly seen for mixed feedlot manure, so impacts on full scale AD should be minimal. Antibiotic degradation products were found, confirming AD effectively degraded ampicillin, florfenicol, and tylosin, although some products were persistent throughout the process. Contamination of AD solid and liquid effluents with sulfamethazine and antibiotic transformation products from florfenicol and tylosin could present an environmental concern., (Published by Elsevier Ltd.)

In this study, the cytochrome P450 3A (CYP3A) gene was cloned from the turbot Scophthalmus maximus for the first time using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches. The amino acid sequences were analyzed with corresponding software programs. The cDNA of CYP3A was 1,969 bp in length, which contained a 5'-untranslated region (UTR) of 34 bp, a 3'-UTR of 404 bp and an open reading frame of 1,530 bp encoding a predicted protein of 509 amino acids (GenBank accession No. JN216889). The deduced protein had a molecular weight of 58.09 kDa and an isoelectric point of 5.75. Amino acid sequence alignment indicated that turbot CYP3A shared 60-67% homology with other fish species. It consists of a signal peptide, six conservative substrate recognition sites (SRS 1-6) and the conserved heme-binding motif FXXGXXXCXG in all CYP3As. Quantitative real-time RT-PCR analysis indicated that turbot CYP3A mRNA was widely expressed in liver, kidney, gill, muscle, stomach, intestine, gallbladder and spleen, with the highest level in liver and the lowest in muscle. After oral administration of sulfamethazine, CYP3A expression in all experimental groups enhanced compared with control, and the expression varied with administration time. It suggested that CYP3A expression could be induced by sulfamethazine. Our findings provided molecular characterization and expression profile of turbot CYP3A, and revealed the important role that turbot CYP3A played in drug metabolisms.

The present study was conducted to evaluate the efficacy of two prophylactic antibiotic treatments against bovine respiratory disease (BRD) in veal calves. In addition, the antibiotic susceptibilities of isolated Pasteurellaceae were tested. The calves were treated either on the day of arrival by a single administration of tulathromycin (group A, n=20), by a peroral administration of chlortetracycline, sulphadimidine, and tylosin (group B, n=20) for seven consecutive days, or were not prophylactically treated (group C, n=19). On the first day of clinically diagnosed BRD, transtracheal lavage samples were obtained prior to therapeutic treatment and were subsequently cultured. Pasteurellaceae isolates were tested for their susceptibility to 12 antimicrobial agents by the determination of the minimal inhibitory concentrations. During the first 56 d after arrival, different calves in group A and B suffered from one episode of clinically diagnosed BRD while calves of group C experienced two episodes. The average daily weight gain during the same period was significantly lower in group C (0.89 ± 0.04kg/d) than in the two prophylactically treated groups (1.14 ± 0.05 and 1.15 ± 0.04 kg/d for group A and B, respectively). The improved performance of groups A and B in comparison to group C could be related to a lower incidence of respiratory disorders during the first days after arrival in the prophylactically treated animals. No differences in the clinical efficacy were seen between the two tested prophylactic treatments. The most prevalent bacterial pathogens isolated (n=79) were Pasteurella multocida (23% of isolated pathogens), Mycoplasma bovis (18%), and Mannheimia varigena (16%). For the isolated Pasteurellaceae, a high resistance pattern was observed to tylosin (83% of the tested P. multocida and 88% of the Mannheimia spp. isolates resistant) and tilmicosin (56% of the tested P. multocida isolates non-sensitive)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic regulation is very limited. Using the Arabidopsis thaliana repressor of silencing1 mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation.

Unlabelled: Antibiotics are a cost-effective tool for improving feed efficiency and preventing disease in agricultural animals, but the full scope of their collateral effects is not understood. Antibiotics have been shown to mediate gene transfer by inducing prophages in certain bacterial strains; therefore, one collateral effect could be prophage induction in the gut microbiome at large. Here we used metagenomics to evaluate the effect of two antibiotics in feed (carbadox and ASP250 [chlortetracycline, sulfamethazine, and penicillin]) on swine intestinal phage metagenomes (viromes). We also monitored the bacterial communities using 16S rRNA gene sequencing. ASP250, but not carbadox, caused significant population shifts in both the phage and bacterial communities. Antibiotic resistance genes, such as multidrug resistance efflux pumps, were identified in the viromes, but in-feed antibiotics caused no significant changes in their abundance. The abundance of phage integrase-encoding genes was significantly increased in the viromes of medicated swine over that in the viromes of nonmedicated swine, demonstrating the induction of prophages with antibiotic treatment. Phage-bacterium population dynamics were also examined. We observed a decrease in the relative abundance of Streptococcus bacteria (prey) when Streptococcus phages (predators) were abundant, supporting the "kill-the-winner" ecological model of population dynamics in the swine fecal microbiome. The data show that gut ecosystem dynamics are influenced by phages and that prophage induction is a collateral effect of in-feed antibiotics., Importance: This study advances our knowledge of the collateral effects of in-feed antibiotics at a time in which the widespread use of "growth-promoting" antibiotics in agriculture is under scrutiny. Using comparative metagenomics, we show that prophages are induced by in-feed antibiotics in swine fecal microbiomes and that antibiotic resistance genes were detected in most viromes. This suggests that in-feed antibiotics are contributing to phage-mediated gene transfer, potentially of antibiotic resistance genes, in the swine gut. Additionally, the so-called "kill-the-winner" model of phage-bacterium population dynamics has been shown in aquatic ecosystems but met with conflicting evidence in gut ecosystems. The data support the idea that swine fecal Streptococcus bacteria and their phages follow the kill-the-winner model. Understanding the role of phages in gut microbial ecology is an essential component of the antibiotic resistance problem and of developing potential mitigation strategies.

Aims: To characterize class 1 integrons and resistance genes in tetracycline-resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control)., Methods and Results: Tetracycline-resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays., Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline-resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline-resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons., Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline-resistant E. coli isolated from feedlot cattle., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Background: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA., Results: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments., Conclusion: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Background: The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. New classes of inhibitors are essential to combat swine influenza viral infection., Methods: A series of isatine-sulfadimidine derivatives were screened for antiviral activity against swine influenza A/California/07/2009 (H1N1) virus in Madin-Darby canine kidney (MDCK) cell culture. Cytotoxicity of the synthesized compounds was also tested in uninfected MDCK cells., Results: All the compounds inhibit the influenza A (H1N1) in MDCK cells. The most active compounds, SPIII-5Br and SPIII-5H, inhibited virus-induced cytopathology by 50% at 27 and 30 microM, respectively, with 50% cytotoxicity occurring at a much higher dose (975-1,000 microM). The positive control compound ribavirin inhibits the replication of the virus at 18 microM and cytotoxic concentration was found to be >1,000 microM., Conclusions: SPIII-5Br and SPIII-5H exhibited potency in the same range as ribavirin and are suitable candidate molecules for further investigation.

The effect of traces of sulfamethazine (SMZ) in soil (0.01, 0.1, 0.25, 1, 5, 15, and 20 mM) on cellular distribution of cytochrome c oxidase activity, shoot and root growth, and leachate electroconductivity was analyzed in germinating seeds of yellow lupin, pea, lentil, soybean, adzuki bean, and alfalfa. Results showed that a high activity of cytochrome c oxidase in mitochondria correlated with high seed vigor and viability. The appearance of necroses and root decay was associated with a decrease in the activity of mitochondrial cytochrome c oxidase but was accompanied by an increase in cytosolic cytochrome c oxidase activity. A short exposure period of seeds (3 and 6 d) to sulfamethazine did not influence germination. Elongation of roots and stems was more sensitive than germination rate as an indicator of soil contamination by sulfamethazine. Among all tested leguminous plants, yellow lupin was the most reliable bioindicator of SMZ contaminated soil.

Unlabelled: The aim of this study was to develop a new selective medium for isolation of Streptococcus anginosus group (SAG) strains, by adding sulphamethazine and aztreonam as selective agents at Mitis-Salivarius agar (MSA), the medium commonly used for recovery of oral streptococci from oral samples., Material and Method: The evaluation of Mitis-Salivarius--sulphamethazine--aztreonam agar (MSSAA) for SAG selectivity was performed by testing the growth of type strains and laboratory-stored clinical isolates of different oral streptococcal species on this medium and also by investigating the SAG recovery on MSSAA in comparison with MSA and the growth inhibition of non-SAG strains from 100 saliva and 11 pus samples (collected from healthy young subjects and from paediatric patients with upper respiratory tract infections, respectively) on MSSAA., Results: The same SAG recovery rate was obtained on both MSSAA and MSA, while non-SAG strains failed to grow on the novel medium, except for enterococci. The results of the present study have indicated that MSSAA is a suitable medium for selecting SAG isolates from clinical specimens.

Background: Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes., Methodology/principal Findings: In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis., Conclusions/significance: The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

In several avian species long day exposure results in plasma elevation of gonadotropins and prolactin (PRL). We examined the early (12-72h) effects of photostimulation on mRNA transcripts and plasma levels of follicle stimulating hormone (FSH) and PRL in three-week old cockerels. In addition, the neuroendocrine influence of the compound, sulfamethazine (SMZ), known to enhance light-induced gonadal development in chicks, was studied when applied with or without long-day photostimulation. Both long day exposure and SMZ intake caused a rapid increase in FSHbeta mRNA transcripts at Zeitgeber time 48 (ZT48), while only SMZ stimulated secretion of the hormone into plasma during the course of the study. In contrast to SMZ treatment, photostimulation was more effective at stimulating PRL mRNA transcripts and secretion of PRL. Results demonstrate a differential role of long day exposure and SMZ intake on the regulation of FSH and PRL synthesis and secretion and suggest that some effects of SMZ on gonadal development may be mediated by the pituitary.

Neurons located in the lateral septal organ (LSO) and medial basal hypothalamus (MBH) have been proposed to be encephalic photoreceptors (EPRs), which sense photoperiodic time and initiate avian gonadal development. Controversy continues regarding the location of EPRs serving the PNES and their signal transduction pathway. Using quantitative real-time RT-PCR we determined activation of key genes following prolonged light periods and sulfamethethazine (compound known to advance light-induced testes development) in 21-day old chicks. Earliest activation occurred in genes of vasoactive intestinal polypeptide (VIP) and type 6 phosphodiesterase beta subunit (PDE-6 beta) in the LSO at 4 and 6h, respectively, after onset of light and sulfamethazine intake. In contrast, no change was detected in the MBH during the first 8h of that treatment. Thereafter, significant increases in gonadotropin releasing hormone (GnRH-1) and VIP receptor (VIPR) mRNA transcripts were detected in the bed nucleus of the pallial commissure (NCPa). Hours later, activation of all four genes (VIP, PDE-6 beta, GnRH-1, VIPR) were induced solely by photostimulation. Deiodinase 2 and tyrosine hydroxylase in the MBH did not show increased gene expression until 12h of photostimulation. Prolactin mRNA transcripts showed significant increases at 4h due to SMZ intake and at 24, 36 and 48 h due to long-day photoperiodic effects. Data suggest that VIP neurons in the LSO may serve as EPRs and utilize PDE, present in the phototransduction cascade of known photoreceptors. Additionally, VIP released from the LSO may modulate GnRH-1 neurons in the NCPa via VIP receptors by increasing GnRH-1 gene expression.

This study was conducted on 60 ovariectomized bitches. The objectives were to measure the mean reactive oxygen species (ROS) concentrations before, during and after surgery, and to investigate the effect of the administration of five different antibiotic treatments: amoxicillin, benzylpenicillin/dihydrostreptomycin, sulfametazine/sulfamerazine/sulfathiazole, enrofloxacin, lincomycin/spectinomycin. The first value recorded represented the mean ROS concentration in anestral bitches and constitutes a reference level with which to compare the subsequent measurements. After premedication, induction of anesthesia and during maintenance and surgery, ROS serum concentrations showed constant values until the end of surgery. After surgery and during antibiotic administration, an increase in ROS concentration occurred, which differed among the five groups in relation to the antibiotics employed. The lowest increases occurred in the groups treated with the combination of lincomycin/spectinomycin, and with amoxicillin; whereas the highest increases were detected in the group treated with enrofloxacin. The three other antibiotics showed an intermediate level of influence on oxidative status.

Objectives: Human N-acetyltransferase 2 (NAT2) genetic polymorphisms may modify drug efficacy and toxicity and cancer susceptibility from carcinogen exposure. Two human NAT2 alleles, NAT2*5I and NAT2*12D, were identified recently. In NAT2*5I, a new single nucleotide polymorphism AT (L137F) was found coexisting with single nucleotide polymorphisms TC (I114T), CT (silent) and AG (K268R). The other allele NAT2*12D consists of a new single nucleotide polymorphism GA (D122N) together with AG (K268R). We undertook a study to characterize these new single nucleotide polymorphisms and NAT2 alleles to further our understanding of genotype/phenotype relationships in human populations., Methods: Various human NAT2 alleles were cloned and recombinantly expressed in COS-1 cells and the effects of single nucleotide polymorphisms on NAT2 expression were determined. To further test our hypothesis that AT (L137F) and GA (D122N) accelerate protein degradation, various NAT2 alleles were cloned and expressed in Escherichia coli, which does not possess the ubiquitin-mediated degradation pathway., Results: Both AT and GA reduced NAT2 immuno-reactive protein to an undetectable level without causing changes in mRNA level. Missense mutants displayed different effects on sulfamethazine N-acetylation activity for both L137 (wild-type: 70.2+/-5.2 nmol/min/mg; L137F: 1.34+/-0.03 nmol/min/mg; L137W: nondetectable; L137I: 34.2+/-2.0 nmol/min/mg; L137G: 0.52+/-0.04 nmol/min/mg) and D122 (wild-type: 70.2+/-5.2 nmol/min/mg; D122R: non-detectable; D122Q: non-detectable; D122E: 1.72+/-0.24 nmol/min/mg). In contrast to the expression in mammalian cells, recombinant NAT2 possessing either of these two single nucleotide polymorphisms showed no reduction in immuno-reactive NAT2 level when expressed in E. coli., Conclusions: These findings suggest that both AT (L137F) and GA (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D.

The photoneuroendocrine system (PNES) of chicks was activated by transferring birds to a long photoperiod and by giving them a diet supplemented with sulfamethazine (SMZ), a compound that augments the effect of long-day photostimulation. We wished to determine (1) the number of gonadotropin-releasing hormone-1 (GnRH-1) neurons in each identified nucleus (n.) in the subpallium and diencephalon and the major terminal fields (TFs) of GnRH-1 neurons, and (2) the effect of SMZ on the immunoreactive expression of GnRH-1 in perikarya. Four groups of birds were exposed to one of two light treatments, viz., light:dark (LD) cycles of LD20:4 or LD8:16, and given one of two rations, viz., control or one supplemented with SMZ (n=5/treatment). After 3 days, chicks were anesthetized, and their brains were prepared for immunocytochemistry with an antibody identifying GnRH-1 neurons. Seven areas or nuclei contained GnRH-1 neurons: paramedial septal n., preoptic periventricular n./periventricular hypothalamic n., bed n. of the pallial commissure (NCPa), parvocellular lateral and medial septal n., lateral septum near the ventral horn of the lateral ventricle, parvocellular lateral anterior thalamic n., and displaced thalamic neurons. Six TFs of GnRH neurons were found including the organum vasculosum of lamina terminalis (OVLT), preoptic recess (POR), hypothalamic recess (HR), lateral septum adjacent to the ventral horn of the lateral ventricle (SL-VLvh) associated with the choroid plexus, subseptal organ (SSO), and external zone of the median eminence. The extensive TFs for GnRH-1 neurons in the OVLT, POR/HR, SL-VLvh, and SSO suggested that a large amount of the peptide was secreted into the ventricular system. The NCPa responded to the photoperiod and SMZ treatments combined, with a significant increase in GnRH-1 cell number compared with birds fed control diets and exposed to a short-day photoperiod. More than 73% of GnRH-1 neurons resided in the septal region of the subpallium and not in the preoptic hypothalamic region characteristic of several mammalian species. Thus, instead of the traditional descriptor hypothalamo-pituitary-gonadal axis, either the septal- or subpallial-pituitary-gonadal axis may be more appropriate for describing the neuroendocrine axis related to gonadal function in birds.

Recent monitoring investigations have shown that antimicrobial agents used in veterinary medicine can cause non-point source contamination of soils through manure spreading. In the present study, the effect of the antimicrobial agent sulfamethazine (sulfadimidine) on degradation and sorption of the herbicide metolachlor in a sandy loam soil was studied. In soil samples treated with sulfamethazine at two concentrations (15 and 150 microg kg(-1) soil), metolachlor persistence was not different than of that observed in untreated samples. These results were supported by the absence of effects of both sulfamethazine concentration levels on the size of the culturable soil bacteria population. Equilibrating soil samples with metolachlor solutions containing equivalent sulfamethazine concentrations did not lead to any significant effects on metolachlor sorption, suggesting that, under the conditions of the present experiment, sulfamethazine did not affect metolachlor bioavailability in soil. This laboratory investigation showed that concentrations of sulfamethazine in the microg kg(-1) range did not cause significant effects on metolachlor degradation and sorption thus not affecting the main processes ruling its environmental fate in soil.

Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease.

The objective of this experiment was to compare the effects of dietary mannan oligosaccharide (MOS) and a feed-grade antimicrobial (AM) on growth performance of nursery pigs reared on three different farms (A and B were large-scale commercial farms, and C was located at Michigan State University). On all farms, production was continuous flow by building, but all-in/all-out by room. Within each nursery facility, all pigs on the experiment were in one room. Pigs (Farm A, n = 771, weaning age = 18.4 d; Farm B, n = 576, weaning age = 19.0 d; Farm C, n = 96, weaning age = 20.6 d) were blocked (within farm) by BW and sex and allotted randomly to dietary treatments arranged in a 2 x 2 factorial. The two factors were 1) with and without MOS (0.3% in Phase I, 0.2% in Phases II, III, and IV; as-fed basis) and 2) with and without AM (110 mg of tylosin and 110 mg of sulfamethazine/kg of diet in all phases; as-fed basis). The four nursery phases were 4, 7, 14, and 17 d, respectively. With 35, 20, and 4 pigs per pen on Farms A, B, and C, respectively, space allowances per pig were 0.29, 0.26, and 0.56 m2. Across all farms, the addition of AM and MOS plus AM increased (P < 0.05) ADG (368, 406, and 410 g/d for control, AM, and MOS plus AM, respectively and increased ADFI (661, 703, and 710 g/d for control, AM, and MOS plus AM, respectively) for the entire 42-d experiment. The addition of MOS also increased ADG (P < 0.05) from d 0 to 42 of the experiment (394 g/d). Performance differed depending on farm (P < 0.01). Antimicrobial did not affect growth performance on Farm B, but it increased (P < 0.05) ADG on Farms A and C, ADFI on Farm A, and G:F on Farm C. Growth improvements with MOS on Farms A and B were not significant; however, pigs on Farm C fed MOS had greater (P < 0.05) ADG, ADFI, and G:F than controls. The results of this study suggest that MOS may be an alternative to tylosin and sulfa-methazine as a growth promotant in nursery diets.

The objective of this study was to determine the effects of sub-minimum inhibitory concentrations (sub-MICs) of 2 veterinary antibiotic preparations, chlortetracycline (CTC) and chlortetracycline-sulfamethazine (CTC + SMZ), on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus at normal and febrile body temperatures. Sub-minimum inhibitory concentrations of both antibiotics reduced the growth rates of M. haemolytica and H. somnus. Growth of both species was not inhibited when grown at 41 degrees C compared to 37 degrees C. There was no detectable consistent effect of antibiotic or temperature on outer membrane protein expression for either species. Our study indicates that sub-MIC levels of CTC and CTC + SMZ markedly impair growth of clinical M. haemolytica and H. somnus isolates, potentially allowing more effective host clearance during infection.

The aims were to provide data on antibiotic residues in pig tissues and the trends in the occurrence of antibiotic resistance in Escherichia coli (a commensal bacteria that can colonize the intestinal tract of humans and animals) isolated from the animals studied. Muscle and kidney samples collected from freshly slaughtered pigs were tested for the presence of antibiotic residues using rapid screening tests. Ninety muscle and 90 kidney samples from the same animals, collected in the slaughterhouse of the central region of Portugal, were screened by microbial tests and only six samples did not fulfil the European Community regulations concerning maximum residue limits. When a more sensitive method was used for the detection of sulphamethazine, 36% of the animals contained residues of the antibiotic. This indication of a larger antibiotic occurrence than anticipated was confirmed by studying the susceptibility of their enteric E. coli. In all the animal faeces studied (n = 60), E. coli isolates resistant to amoxycillin, tetracyclines and sulphonamides were found. The majority of the faecal samples demonstrated a high degree of E. coli resistant to sulphonamides and tetracyclines, respectively 88% and 85%. These data raise important questions about the relevance of the detection of antibiotic residues as the only method to establish the quality and safety of animal products for human consumption.

A sulfonamide, sulfamethazine (SMZ) has been shown to have a robust, progonadal effect. The mechanism of action of SMZ, however, is unknown. Our hypothesis is that the compound may act centrally and/or at the level of the pituitary. Four experiments were completed to test that hypothesis. Chicks exposed to a continuous photoperiod and fed a diet containing 0.2% SMZ showed an exponential increase in testes size. When 6 weeks of age (5 weeks on the SMZ diet), experimentals had testes weight nine times heavier than controls. Profiles for thyroid and gonadotropin plasma hormones suggested that T(3) was transiently lower in experimentals solely during the first week on treatment, while thyroxine levels were not different from controls. In contrast, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly elevated at the initial 1-week sampling point and remained elevated throughout the entire experiment. In a follow-up study, LH was found significantly higher than controls by 48 h after initially consuming the compound. When T(3) was added to the SMZ diet at 0.5 ppm, the progonadal effect of SMZ was attenuated. Importantly, chronic intake of T(3) delayed but did not block the stimulatory effect of SMZ for increasing plasma LH. We conclude that since one of the primary effects of SMZ is to increase rapidly plasma gonadotropins, data suggest the compound is acting at the level of the brain or pituitary to stimulate early gonadal development in chicks.

Arylamine N-acetyltransferase (NAT) enzymes catalyze the addition of an acetyl group from acetyl-CoA to a terminal nitrogen on a suitable substrate such as environmentally relevant compounds and pharmaceuticals. In human, there are two highly polymorphic active allozymes, NAT1 and -2, and one inactive pseudogene, NATP. The expression of these enzymes is tissue-specific such that NAT1 is ubiquitously expressed and NAT2 is confined mainly to liver and colorectal tissues. We hypothesized that these genes would be tissue-specifically transcriptionally regulated, and so we isolated putative proximal control regions for both the NAT genes, which were inserted into luciferase vectors and transiently transfected into human liver and bladder cells. The transfected cells were dosed with 4-aminosalicylic acid, sulfamethazine or solvent and the resulting luciferase activity was measured. We found that both NAT1 and -2 regions were inducible in liver cells by both xenobiotics but only one of the NAT1 regions was inducible again by both xenobiotics in bladder cells. These results suggest that the NAT genes may be tissue-specifically transcriptionally regulated.

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its derivatives were synthesized by reaction of isatin and its derivatives with sulphadimidine. Their chemical structures have been confirmed by IR, (1)H NMR data and elemental analysis. Investigation of anti-HIV activity of compounds were tested against replication of HIV-1 (IIIB) and HIV-2 (ROD) strains in acutely infected MT-4 cells and the activity compared with standard azidothymidine. Among the compounds tested, 4-[(1,2-dihydro-2 oxo-3H-indol-3-ylidene)amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its N-acetyl derivative were the most active compounds.

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.

Skin is the major target of allergic reactions to paraphenylenediamine (PPD). Such small molecules require activation to become immunogenic. The balance between activation and/or detoxification processes is critical for immunogenic potentials of compounds. Therefore, we investigated N-acetylation (NAT) capacities of human skin for PPD to gain a better understanding of its mechanisms of action. PPD is acetylated to monoacetyl-PPD (MAPPD), which in turn is acetylated to N,N'-diacetyl-PPD (DAPPD). This was found using cytosolic fractions from human skin (n = 9) and cultured normal human epidermal keratinocytes (n = 7). The cutaneous activities for MAPPD formation ranged from 0.41 to 3.68 nmol/mg/min (9-fold variation) and DAPPD formation from 0.65 to 3.25 nmol/mg protein/min (5-fold), respectively. Similar results were obtained with keratinocytes. NAT activities toward both substrates, PPD and MAPPD, were correlated in keratinocytes (r = 0.930), suggesting that the reactions were catalyzed by the same enzyme. Formation of MAPPD and DAPPD was competitively inhibited in the presence of p-aminobenzoic acid (300 microM), a typical NAT1 substrate, but not by sulfamethazine. These kinetic characteristics suggest that the acetylation of PPD in human skin and keratinocytes is predominantly attributable to the polymorphic NAT1, although both mRNAs (NAT1 and NAT2) are synthesized in human skin and keratinocytes. The metabolism of PPD by NAT1 in human skin and keratinocytes as well as the virtual absence of NAT2 activity may have important toxicological implications. In the case of PPD, our results emphasize that N-acetylation status may be a susceptibility factor for the development of an allergy to PPD.

This study was conducted to determine whether an antimicrobially induced (ASP-250) increase in serum IGF-I was the result of differences in feed intake. Serum IGF-I concentrations were measured in crossbred pigs that were fed a control diet or a diet supplemented with ASP-250 either for ad libitum consumption or limited to 85% of the control pigs' consumption. The pigs that consumed either diet ad libitum, control or ASP-250, consumed similar quantities of feed. The ASP-250 ad libitum-intake pigs had serum IGF-I concentrations that were greater (P<.01) than those of their ad libitum-intake control littermates. Similarly, the ASP-250 limit-fed pigs had serum IGF-I concentrations that were greater (P<.01) than those of the controls. Although the serum IGF-I concentrations of pigs fed the ASP-250-supplemented diet for ad libitum intake were greater than the serum IGF-I concentrations of the pigs limit-fed the ASP-250-supplemented diet, the differences were not significant (P<.08). The ASP-250-fed pigs had higher serum IGF binding protein (BP)-3 concentrations than did their control littermates (P<.003). A time course of antimicrobially induced alterations in serum IGF-I concentrations revealed that the effect of increased serum IGF-I levels in ASP-250-supplemented pigs (P<.02) was observed within 4 d and was maintained throughout the 4-wk study. These findings show that feed intake is not responsible for the increase in serum IGF-I observed with ASP-250 supplementation. Additionally, the antimicrobially induced increase in serum IGF-I concentrations occurs within a few days after initiation of the treatment.

A significant sex-related difference was observed for the pharmacokinetics of acetohexamide in Wistar-Imamichi (Wistar-IM) rats. However, there was no sex difference of the in vitro reductive metabolism of acetohexamide in the liver or kidney of these rats. Testectomy was found to decrease the plasma clearance (CLp) of acetohexamide in male rats, whereas ovariectomy had no effect on the CLp of acetohexamide in female animals, suggesting that androgens regulate the pharmacokinetics of acetohexamide. The co-administration of sulfamethazine, which is known to be metabolized by a male-specific cytochrome P450 (CYP) isoform (CYP2C11), significantly decreased the CLp of acetohexamide in male Wistar-IM rats. Based on these results, it is reasonable to assume that the sex-dependent pharmacokinetics of acetohexamide observed in Wistar-IM rats is associated with the male-specific hydroxylation catalyzed by CYP2C11.

Two human acetyl-CoA:arylamine N-acetyltransferases (NAT1 and NAT2) have been identified. Therapeutic and carcinogenic agents that are substrates for these isoenzymes (including isoniazid, sulfamethazine, p-aminobenzoic acid, 5-aminosalicyclic acid, and 2-aminofluorene) have been used to evaluate the role of the N-acetylation polymorphisms of NAT1 and NAT2 in the treatment of disease and differential risk of various cancers among individuals of differing acetylator phenotypes. The mouse is frequently used as a model of the human acetylator polymorphism. As three Nat isoenzymes have been identified in mouse, it is necessary to determine the selectivity of mouse Nats toward common NAT substrates. In the present study, Nat1*, Nat2*8, and Nat3* were expressed in COS-1 cells, and their substrate selectivity was evaluated with various substrates. Under the conditions used, mouse Nat2 had 20-, 2.4-, and 5.4-fold higher catalytic activity for p-aminobenzoic acid, 5-aminosalicylic acid, and 2-aminofluorene, respectively, than Nat1. Isoniazid N-acetylation was catalyzed only by mouse Nat1. For the substrates tested in this study, mouse Nat3 exhibited activity only toward 5-aminosalicylic acid and only at 1/20 the activity shown by Nat2. In addition, p-aminobenzoylglutamate, the first endogenous NAT substrate identified, was selective for mouse Nat2. These results further support the functional analogy of mouse Nat2 and human NAT1.

Twelve calves aged 6-10 months, and 12 calves aged 10-16 months were turned out onto a permanent pasture known to have been contaminated with oocysts of Eimeria alabamensis during the previous year. Two days after turnout, six of the older calves and six of the younger were each treated with one bolus per 200 kg bodyweight containing 1.6 g baquiloprim and 14.4 g sulphadimidine. The other 12 calves were left untreated. The excretion of Eimeria oocysts, the faecal dry matter and the weight gain of treated and untreated calves within each age group were compared during the first 3 weeks on pasture to assess the efficacy of the bolus in preventing E. alabamensis coccidiosis. All the older of the untreated calves and four of the younger developed gruel-like to watery diarrhoea 4-7 days after turnout. The faecal consistency of the treated calves remained firm and they lost significantly less weight than the control calves during the first 13 days on pasture. The treated calves also excreted significantly fewer oocysts during the first 20 days of grazing; their oocyst excretion remained low during days 8-10 when all but one of the diarrhoeic control calves excreted more than 850,000 oocysts per gram faeces (OPG). Starting on days 12 to 14 the oocyst excretion of 8 of the treated calves increased to 20,000-65,000 OPG and of 2 calves to 210,000-240,000 OPG. There was no difference in oocyst output between treated and untreated calves from the fourth week of grazing and no difference in weight gain among the younger calves. In the older calves there was a tendency for the untreated calves to gain more weight than treated calves.

Sulfamethazine (SMZ) administration (ip, 3 d) of different doses to adult male rats showed significant increases in the electron transport components and activities of aminopyrine N-demethylase and aniline hydroxylase at the the 150 mg SMZ/kg dose level. However, 300 mg SMZ/kg doses produced a significant decrease in cytochrome P-450 and the activity of aminopyrine N-demethylase. In a longer duration study, 300 mg SMZ/kg caused significant decreases in cytochrome P-450 and in the activity of aminopyrine N-demethylase. The inducer dose of 150 mg SMZ/kg and the inhibitory dose of 300 mg SMZ/kg were selected for dosing young male, old male and adult female rats. Sulfamethazine administration to young male rats resulted in a significant induction of electron transport components and drug metabolizing enzymes at both dose levels. However, SMZ treatment of old rats produced significant decreases in electron transport components and aminopryine N-demethylase activity at both dose levels. A significant induction in the levels of electron transport components was observed with 150 mg SMZ/kg in female rats. All other parameters were unchanged. Sulfamethazine resulted in a mixed type of inhibition (Ki = 3.5 mM) of aminopyrine N-demethylase in vitro. Hydroxylated metabolites destructed the spectral and catalytic activity of cytochrome P-450. Our studies suggest that SMZ is a substrate of the mixed function oxidase system and induction is dependent on dosage, age and sex of the animals.