Search

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysR ars , which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysR ars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysR ars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysR ars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysR ars , respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The organoarsenical feed additive roxarsone (ROX) is a ubiquitous threat due to the unpredictable levels of arsenic (As) released by soil bacteria. The earthworms representing soil fauna communities provide hotspots for As biotransformation genes (ABGs). Nonetheless, the role of gut bacteria in this regard is unclear. In this study, the changes in As speciation, bacterial ABGs, and communities were analyzed in a ROX-contaminated soil (50 mg/kg As in ROX form) containing the earthworm Eisenia feotida. (RE vs. R treatment). After 56 d, earthworms reduced the levels of both ROX and total As by 59 % and 17 %, respectively. The available As content was 10 % lower in the RE than in R treatment. Under ROX stress, the total ABG abundance was upregulated in both earthworm gut and soil, with synergistic effects observed following RE treatment. Besides, the enrichment of arsM and arsB genes in earthworm gut suggested that gut bacteria may facilitate As removal by enhancing As methylation and transport function in soil. However, the bacteria carrying ABGs were not associated with the ABG abundance in earthworm gut indicating the unique strategies of earthworm gut bacteria compared with soil bacteria due to different microenvironments. Based on a well-fit structural equation model (P = 0.120), we concluded that gut bacteria indirectly contribute to ROX transformation and As detoxification by modifying soil ABGs. The positive findings of earthworm-induced ROX transformation shed light on the role of As biomonitoring and bioremediation in organoarsenical-contaminated environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hepatic fibrosis is a pathological change caused by chronic liver injury and self-repair, and it is the inevitable stage of the development of chronic liver disease to cirrhosis or even liver cancer. Activation of hepatic stellate cells (HSCs) is a core event in the development of liver fibrosis and blockage of the activation of HSCs has been shown to alleviate liver fibrosis. Roxarsone, an organoarsenic additive, with antibiotic effect, growth promotion and improving feed efficiency, is widely used in livestock and animal production. The purpose of this study was to evaluate the therapeutic effect of Roxarsone on liver fibrosis and explore the possible mechanism. We found that Roxarsone could inhibit transforming growth factor-β1 (TGF-β1) induced the activation of HSCs and weaken the migration ability. Moreover, Roxarsone administration significantly ameliorated CCl 4 -induced liver fibrosis in mice with improvement of liver function and decreases of deposition of extracellular matrix (ECM). Mechanism investigations revealed that Roxarsone specifically inhibited the activation of TGF-β1/Smad signaling pathway, but had no effect on MAPK and PI3K/AKT pathways. These results suggest that Roxarsone has a protective effect on liver fibrosis which provides a new candidate for the treatment of liver fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance ( ars ) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE , a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE , cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).

In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarR ars . MarR ars orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarR ars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV . Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarR ars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarR ars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarR ars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarR ars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarR ars , regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarR ars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.

The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1-50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12-48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

Arsenic-resistant bacteria have evolved various efflux systems for arsenic resistance. Five arsenic efflux proteins, ArsB, Acr3, ArsP, ArsJ, and MSF1, have been reported. In this study, comprehensive analyses were performed to study the function of a putative major facilitator superfamily gene, arsK , and the regulation of arsK transcriptional expression in Agrobacterium tumefaciens GW4. We found that (i) arsK is located on an arsenic gene island in strain GW4. ArsK orthologs are widely distributed in arsenic-resistant bacteria and are phylogenetically divergent from the five reported arsenic efflux proteins, indicating that it may be a novel arsenic efflux transporter. (ii) Reporter gene assays showed that the expression of arsK was induced by arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], methylarsenite [MAs(III)], and arsenate [As(V)]. (iii) Heterologous expression of ArsK in an arsenic-hypersensitive Escherichia coli strain showed that ArsK was essential for resistance to As(III), Sb(III), Rox(III), and MAs(III) but not to As(V), dimethylarsenite [dimethyl-As(III)], or Cd(II). (iv) ArsK reduced the cellular accumulation of As(III), Sb(III), Rox(III), and MAs(III) but not to As(V) or dimethyl-As(III). (v) A putative arsenic regulator gene arsR2 was cotranscribed with arsK , and (vi) ArsR2 interacted with the arsR2-arsK promoter region without metalloids and was derepressed by As(III), Sb(III), Rox(III), and MAs(III), indicating the repression activity of ArsR2 for the transcription of arsK These results demonstrate that ArsK is a novel arsenic efflux protein for As(III), Sb(III), Rox(III), and MAs(III) and is regulated by ArsR2. Bacteria use the arsR2-arsK operon for resistance to several trivalent arsenicals or antimonials. IMPORTANCE The metalloid extrusion systems are very important bacterial resistance mechanisms. Each of the previously reported ArsB, Acr3, ArsP, ArsJ, and MSF1 transport proteins conferred only inorganic or organic arsenic/antimony resistance. In contrast, ArsK confers resistance to several inorganic and organic trivalent arsenicals and antimonials. The identification of the novel efflux transporter ArsK enriches our understanding of bacterial resistance to trivalent arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], and methylarsenite [MAs(III)]., (Copyright © 2018 American Society for Microbiology.)

Enrofloxacin (ENR) and roxarsone (ROX) have been widely used in animal breeding. In this study, the daily clinical dosage of ENR and daily additive amount of ROX were administrated to Bama pigs. After 5days, the activity and protein expression of three important enzymes in the cytochrome P450 family were measured in the porcine liver. CYP1A2 was induced by both ENR and ROX independently. CYP2E1 and CYP3A4 were inhibited by ENR, but not affected by ROX. The combined administration of ENR and ROX were antagonistic to CYP1A2 and CYP2E1, but not to CYP3A4. Drug-drug interactions should be considered during the administration of ENR, ROX and for their co-administration with other drugs to minimize adverse reactions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Roxarsone (ROX) is an organoarsenic feed additive of increasing interest used in the poultry industry. Soybean responses to ROX stress were investigated in root exudates (REs) using two-dimensional correlation spectroscopy (2D-COS) with fluorescence and Fourier transform infrared spectra. Environmentally relevant ROX concentrations caused negligible toxicity to crop growth and photosynthesis activity but blackened soybean roots at high concentrations. 2D-COS analysis revealed that the protein-like fluorophore and C═C and C═O, aliphatic OH, and polysaccharide C-O-H moieties in soybean REs were most sensitive to ROX stress. Heterospectral 2D-COS results suggested that aromatic, amide I, quinone, ketone, and aliphatic functional groups were the foundational components of protein-like and short-wavelength excited humic-like fluorophores in soybean REs. Carboxyl and phenolic moieties were related to the long-wavelength excited humic-like fluorophore. Overall, 2D-COS combined with molecular-based spectral analysis of REs provided an innovative approach to characterize the physiological responses of crops to contaminants at sublethal levels.

Roxarsone is a feed additive widely used in the broiler and swine industries that has the potential to contaminate the environment, mainly via the use of poultry manure as fertilizer, which results in release of inorganic arsenic to the soil and water. This study was conducted to investigate roxarsone degradation and the response of the microbial community under different culture conditions using high-throughput sequencing technology. Poultry litter was incubated for 288 h in the presence of roxarsone under light aerobic, dark aerobic, or dark anaerobic conditions. The results showed that roxarsone was completely degraded after 48 h of dark anaerobic incubation, while 79.9% and 94.5% of roxarsone was degraded after 288 h of dark aerobic and light aerobic incubation, respectively. Under dark aerobic conditions with microbial inhibitor sodium azide, roxarsone was rarely degraded during the 288 h of incubation, illustrating that microorganisms play an important role in roxarsone degradation. Microbial community structure was significantly different among various culture conditions. Olivibacter, Sphingobacterium, and Proteiniphilum were the top 3 genera in the control samples. Sphingobacterium and Alishewanella dominated the light aerobic samples, while the dominant microflora of the dark aerobic samples were Acinetobacter spp. Pseudomonas and Advenella were the predominant genera of dark anaerobic samples. This study emphasizes the potential importance of microbes in roxarsone degradation and expands our current understanding of microbial ecology during roxarsone degradation under different environmental conditions.

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals., (© 2015 John Wiley & Sons Ltd.)

Environmental organoarsenicals are produced by microorganisms and are introduced anthropogenically as herbicides and antimicrobial growth promoters for poultry and swine. Nearly every prokaryote has an ars (arsenic resistance) operon, and some have an arsH gene encoding an atypical flavodoxin. The role of ArsH in arsenic resistance has been unclear. Here we demonstrate that ArsH is an organoarsenical oxidase that detoxifies trivalent methylated and aromatic arsenicals by oxidation to pentavalent species. Escherichia coli, which does not have an arsH gene, is very sensitive to the trivalent forms of the herbicide monosodium methylarsenate [MSMA or MAs(V)] and antimicrobial growth promoter roxarsone [Rox(V)], as well as to phenylarsenite [PhAs(III), also called phenylarsine oxide or PAO]. Pseudomonas putida has two chromosomally encoded arsH genes and is highly resistant to the trivalent forms of these organoarsenicals. A derivative of P. putida with both arsH genes deleted is sensitive to MAs(III), PhAs(III) or Rox(III). P. putida arsH expressed in E. coli conferred resistance to each trivalent organoarsenical. Cells expressing PpArsH oxidized the trivalent organoarsenicals. PpArsH was purified, and the enzyme in vitro similarly oxidized the trivalent organoarsenicals. These results suggest that ArsH catalyzes a novel biotransformation that confers resistance to environmental methylated and aromatic arsenicals., (© 2015 John Wiley & Sons Ltd.)

Roxarsone is an organoarsenic feed additive which can be finally degraded to other higher toxic metabolites after excreted by animal. In this work, the uptake of As species by vegetables treated with chicken manure bearing roxarsone and its metabolites was investigated. It was showed that more than 96% of roxarsone added in chicken feed was degraded and converted to arsenite, monomethylarsonic acid, dimethylarsinic acid, arsenate, 4-hydroxyphenylarsonic acid and other unknown As species. Arsenite and arsenate could be found in roots of vegetables but only arsenite transported up to shoots. Chicken manure bearing roxarsone and its metabolites increased 33-175% of arsenite and 28% ∼ seven times of arsenate in vegetable roots, 68-175% of arsenite in edible vegetable shoots. Arsenite, the most toxic As form, was the major extractable As species in vegetables accounted for 79-98%. The results reflected that toxic element As could be absorbed by vegetables via the way: roxarsone in feed → animal → animal manure → soil → crop and the uptake of As species would be enhanced by using chicken manure bearing roxarsone and its metabolites as organic fertilizer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The toxic metalloid arsenic is widely distributed in food, water, and soil. While inorganic arsenic enters the environment primarily from geochemical sources, methylarsenicals either result from microbial biotransformation of inorganic arsenic or are introduced anthropogenically. Methylarsenicals such as monosodium methylarsonic acid (MSMA) have been extensively utilized as herbicides, and aromatic arsenicals such as roxarsone (Rox) are used as growth promoters for poultry and swine. Organoarsenicals are degraded to inorganic arsenic. The toxicological effects of arsenicals depend on their oxidation state, chemical composition, and bioavailability. Here we report that the active forms are the trivalent arsenic-containing species. We constructed a whole-cell biosensor utilizing a modified ArsR repressor that is highly selective toward trivalent methyl and aromatic arsenicals, with essentially no response to inorganic arsenic. The biosensor was adapted for in vitro detection of organoarsenicals using fluorescence anisotropy of ArsR-DNA interactions. It detects bacterial biomethylation of inorganic arsenite both in vivo and in vitro with detection limits of 10(-7) M and linearity to 10(-6) M for phenylarsenite and 5 × 10(-6) M for methylarsenite. The biosensor detects reduced forms of MSMA and roxarsone and offers a practical, low cost method for detecting activate forms and breakdown products of organoarsenical herbicides and growth promoters.

Although bacterial mechanisms involved in the resistance to inorganic arsenic are well understood, the molecular basis for organic arsenic resistance has not been described. Campylobacter jejuni, a major food-borne pathogen causing gastroenteritis in humans, is highly prevalent in poultry and is reportedly resistant to the arsenic compound roxarsone (4-hydroxy-3-nitrobenzenearsonic acid), which has been used as a feed additive in the poultry industry for growth promotion. In this study, we report the identification of a novel membrane transporter (named ArsP) that contributes to organic arsenic resistance in Campylobacter. ArsP is predicted to be a membrane permease containing eight transmembrane helices, distinct from other known arsenic transporters. Analysis of multiple C. jejuni isolates from various animal species revealed that the presence of an intact arsP gene is associated with elevated resistance to roxarsone. In addition, inactivation of arsP in C. jejuni resulted in 4- and 8-fold reductions in the MICs of roxarsone and nitarsone, respectively, compared to that for the wild-type strain. Furthermore, cloning of arsP into a C. jejuni strain lacking a functional arsP gene led to 16- and 64-fold increases in the MICs of roxarsone and nitarsone, respectively. Neither mutation nor overexpression of arsP affected the MICs of inorganic arsenic, including arsenite and arsenate, in Campylobacter. Moreover, acquisition of arsP in NCTC 11168 led to accumulation of less roxarsone than the wild-type strain lacking arsP. Together, these results indicate that ArsP functions as an efflux transporter specific for extrusion of organic arsenic and contributes to the resistance to these compounds in C. jejuni.

Addition of roxarsone can significantly improve the growth of broiler chickens (Gallus gallus domesticus). Nevertheless, this application will lead to the contamination of the environment as well as animal products. Understanding the response of genes to roxarsone may bring about the discovery of new, safer substitutes. In this study, we monitored the expression of 8,935 genes in chicken breast muscle using microarrays. Analysis showed that 30 genes, such as the interleukin 3 regulated nuclear factor (NFIL3), the regulatory factor X-associated ankyrin-containing protein (RFXANK), the cleavage and polyadenylation-specific factor 3 (CPSF3), and the FK506 binding protein 9 (FKBP9), have consistently up or downregulated (fold change ≥1.5 or ≤0.6, P < 0.05, false discovery rate ≤0.05) throughout the medication periods. The results from microarray analysis were validated by real-time quantitative PCR. Further functional investigation showed that 13 of the identified genes are well documented, and surprisingly, 11 (85%) of these are related to immunity (5 are immunity and defense related, 4 are immunodeficiency disease related, 2 are immunosuppressive drug related), and the remaining 2 are energy metabolism related. These findings may suggest that supplement of roxarsone can improve the immunity of chickens through regulating the expression of associated genes, and as a result contribute to the growth promotion. Further research on the encoded proteins of the differentially expressed genes should provide more evidence for the potential mechanism.

Roxarsone is a commonly used additive in chicken (Gallus gallus) industry. However, little is known on the intrinsic molecular mechanism via which the growth performance of birds improves. This study was therefore performed to investigate the expression profiles of genes induced by roxarsone. Fifty-six broiler chickens were divided into two groups, namely treated and untreated with roxarsone. The treated group was provided a diet of 45.4mg/kg roxarsone medication and the other group acted as control. Data analysis showed that roxarsone consistently and significantly (P<0.05) increased chicken growth performance. In addition to this a significant (P<0.05) increase of arsenic residue in liver has been seen. Microarray expression analysis of 8935 genes in liver showed that 22 genes (10 up- and 12 down-regulated) had altered expression throughout the experimental periods. Two novel genes (GenBank accession no. GU724343 and GU724344) were cloned through rapid amplification of cDNA ends (RACE). Gene GU724343 was predicted to encode an unidentified protein and the second gene GU724344 was presumed to encode a new member of immunoglobulin-like receptor (CHIR) family. Our results suggested for the first time that the role of roxarsone could be mainly to modify the expression levels of cell growth, immunity/defense and energy metabolism associated genes, as a result promoting animal growth. Further research on these genes should help to increase the knowledge of improving animal productivity safely and effectively., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Salmonella enterica isolates from turkeys in two commercial processing plants (1 and 2) were characterized for susceptibility to antibiotics, disinfectants, and the organoarsenical growth promoter, 4-hydroxy-3-nitrophenylarsonic acid (3-NHPAA, roxarsone), and it's metabolites, NaAsO(2) (As(III)) and Na(2)HAsO(4) • 7H(2)O (As(V)). The 130 Salmonella serovars tested demonstrated a low incidence of resistance to the antibiotics gentamicin (GEN), kanamycin (KAN), sulfamethoxazole (SMX), streptomycin (STR), and tetracycline (TET). Isolates resistant to antibiotics were most often multidrug resistant. Serovars Hadar and Typhimurium were resistant to KAN, STR, and TET and GEN, SMX, and STR, respectively. All isolated Salmonella serovars were resistant to the disinfectant chlorhexidine with minimum inhibitory concentrations (MICs; 1-8 μg/mL), and they were susceptible to triclosan and benzalkonium chloride. The didecyldimethylammonium chloride component was the most active ammonium chloride tested. No cross-resistance was observed between antibiotics and disinfectants. The MICs for 3-NHPAA (4096 μg/mL) were consistent between processing Plant 1 and Plant 2, but MICs for the 3-NHPAA metabolites (As(III) and As(V)) were higher in Plant 1 than in Plant 2. In Plant 1, 76% of the isolates had MICs >256 μg/mL for As(III) and 92% of the isolates had MICs >1024 μg/mL for As(V). In Plant 2, all of the isolates had MICs ≤256 μg/mL for As(III) and 90% of the isolates had MICs ≤1024 μg/mL for As(V). Only 4 Salmonella serovars were isolated from Plant 1, but 10 serovars were isolated from Plant 2. S. enterica serovar Derby from Plant 1 was highly resistant to As(III) and As(V) with MICs >1024 and >8192 μg/mL, respectively, suggesting previous exposure to high arsenic metabolite concentrations. These levels may have been high enough to kill other Salmonella serovars, thus possibly explaining the lack of serovar diversity observed in Plant 1. The application of a growth promoter may affect the serovar diversity in treated birds.

A collection of 125 Salmonella enterica poultry isolates (71 serovar Kentucky isolates, and the remainder belonging to serovars Alachua, Enteritidis, Hadar, Heidelberg, Montevideo, Mbandaka, Senftenberg, Typhimurium, and Worthington) were tested for the ability to grow on tryptic soy agar containing sodium arsenite [As(III)] or arsenate [As(V)]. All serovar Kentucky isolates and 18 of the non-Kentucky isolates were able to grow in the presence of 0.1 mM As(III), and 69 grew in the presence of 1 mM As(V). Thirty of the non-Kentucky isolates did not grow at these As(III) and As(V) concentrations, but seven grew at 1 mM As(III) and 10 mM As(V). PCR-based analysis demonstrated the presence of arsB and arsD sequences in all Kentucky isolates, whereas one or both of these sequences were present in only 30 of the other isolates. It remains to be determined if these arsenic-resistance determinants benefit Salmonella exposed to man-made arsenic-containing compounds in poultry environments.

Clostridial species predominate in both chicken gastrointestinal tract as well as litter where the organoarsenical roxarsone (3-nitro 4-hydroxybenzenearsonic acid) is anaerobically transformed releasing the more recognized toxic inorganic arsenic. 2D-gel electrophoresis and mass spectrometry were used to evaluate the changes in protein expression of Alkaliphilus oremlandii in response to different growth conditions (e.g., terminal electron acceptors) in order to explore the mechanism of microbial biotransformation of roxarsone. Aldehyde ferredoxin oxidoreductase, the enzyme that belongs to the xanthine oxidase family of molybdoenzymes was significantly overexpressed in the presence of roxarsone suggesting a role in the anaerobic metabolism of this substituted nitrophenol.

Organoarsenicals are commonly used for growth promotion in U.S. poultry production. Susceptibilities to arsenite, arsenate, and the organoarsenical roxarsone were measured in 251 Campylobacter isolates from conventional and antimicrobial-free retail poultry products. Isolates from conventional poultry products had significantly higher roxarsone MICs (z = 8.22; P < 0.0001).

The acute acetoclastic methanogenic inhibition of several inorganic and organic arsenicals was assayed. Trivalent species, i.e., methylarsonous acid and arsenite, were highly inhibitory, with 50% inhibitory concentrations of 9.1 and 15.0 microM, respectively, whereas pentavalent species were generally nontoxic. The nitrophenylarsonate derivate, roxarsone, displayed moderate toxicity.

Histomoniasis or blackhead is a life-threatening disease of turkeys that is caused by a flagellated protozoan, Histomonas meleagridis. The development of an assay to measure the sensitivity of drugs traditionally used against this parasite, as reputed to be effective against other protozoan parasites, is described. The in vitro minimum lethal concentrations (MLC), time for drug efficacy, and parasite viability after removal of residual drugs were determined. Three of the 10 tested drugs, fenbendazole, albendazole, and sulfadiazine, were found to be ineffective against H. meleagridis. Nifursol, the only compound still authorized as a feed additive in Europe, is an inhibiting agent but is not lethal in vitro. Roxarsone, an arsenical derivate similar to nitarsone (the only authorized drug in United States), is effective at high concentration (200 microg/mL) after a long exposure (48 h). The lethal activity of dimetridazole, metronidazole, ronidazole, tinidazole, and furazolidone in vitro was demonstrated. Dimetridazole (MLC = 25 microg/mL after 6 h of exposure), metronidazole (MLC = 50 microg/mL after 24 h), and furazolidone (MLC = 50 microg/mL after 24 h) are rapidly effective at low concentrations. These results confirm the effectiveness of dimetridazole, a drug that has been used in the treatment and prevention of blackhead. In May 2002 this compound was removed as feed additive in Europe.

1. The aim of this study was to examine the effects of roxarsone (3-nitro-4-hydroxyphenylarsonic acid) inclusion in the diet on the performance, liver function and lipid metabolism in the liver of laying Brown Tsaiya ducks. 2. Sixty 36-week-old laying ducks were selected and allocated at random into 4 dietary treatments with 3 replications for each treatment. Feeding was for 7 weeks with 3 weeks of experimental diets followed by a 4 week withdrawal period. The experimental diets were supplemented with 0, 50, 100 and 300 mg/kg roxarsone, respectively 3. Dietary inclusion of 50 or 100 mg/kg roxarsone did not significantly promote performance. Inclusion of 300 mg/kg significantly depressed (P<0.05) performance, liver weight and content, serum triacylglycerol (TG), serum nonesterified fatty acid (NEFA) and increased (P<0.05) cholesterol, creatine kinase (CK) and aspartate aminotransferase (AST) in the serum at the end of 3 weeks on the experimental diet. 4. Laying characteristics returned to normal 4 weeks after withdrawal of roxarsone. The liver weight, fat and TG in the liver and serum concentrations of TG, NEFA, high density lipoprotein (HDL) and AST increased significantly (P<0.05), while the level of very low density lipoprotein (VLDL) decreased (P<0.05) at the end of the withdrawal period. More prominent vacuolised hepatic fatty cells were observed in laying ducks treated with 300 mg/kg of roxarsone.

Two 4 x 2 factorial experiments were designed to test the effects of four diets, differing in anticoccidial programs and roxarsone (3-nitro-4-hydroxyphenylarsonic acid) content, and two lighting programs on the incidence of leg abnormalities in 56-d-old male and female broiler chickens. The four diets were: A) basal diet + salinomycin (60.0 g/ton); B) basal diet + salinomycin (60.0 g/ton) + roxarsone (45.4 g/ton); C) basal diet + roxarsone (45.4 g/ton) + live coccidial vaccine; and D) basal diet + live coccidial vaccine. The light treatments were: 1) restricted (R) [18 h light (L): 6 h dark (D)] and 2) standard (S) [23L:1D]. The respective experiments were conducted during winter (Experiment 1, November to January) and summer (Experiment 2, June to August) growing conditions in the southeastern U.S. Light treatment had no significant (P > 0.10) effect on the incidence of leg abnormalities in either experiment. However, in Experiment 1, there was a light treatment by sex interaction; leg abnormalities were greater for males on Treatment R than Treatment S. In Experiment 2, females had a higher incidence of leg abnormalities than males (8.29 vs 4.73%). There was a diet by sex interaction for the incidence of leg abnormalities in Experiment 2; females had a greater incidence of leg abnormalities than males on Treatment A. In both experiments, the incidence was greater with dietary treatments containing roxarsone (P < 0.10). The percentage of leg abnormalities was greater in Experiment 2 than Experiment 1. Leg abnormalities were primarily due to fibrosis of the gastrocnemius tendons and tendon sheaths.

Six trials were conducted at various locations to determine the response of broiler chickens to combinations of zinc bacitracin and roxarsone when fed in the presence of narasin. The addition of zinc bacitracin at 55 mg per kg significantly improved growth rate and feed utilization when data from all locations were combined. There was no effect of roxarsone fed at 50 mg per kg on BW or feed utilization, nor was there an interaction of roxarsone and zinc bacitracin on BW. There was a significant interaction of roxarsone and zinc bacitracin for feed utilization; addition of zinc bacitracin significantly improved feed utilization both in the presence and absence of roxarsone, but the improvement was greater in the absence of roxarsone.

A buffered propionic acid (BPA) was added to broiler diets fed in floor pens with litter. The BPA was fed continuously at 0, .2, .4, and .8% in Trial 1 and at 0 and .4% in Trial 2. The BPA was also fed at .4% for the last 7 days in Trial 2. Natural salmonellae exposure versus periodic dosage with Salmonella typhimurium was compared in Trial 2. In Trial 1, the BPA supplement had no adverse effects on growth, feed utilization, or abdominal fat with a significant (P less than or equal to .05) increase in the female dressing value at .8% of buffered propionic acid. The total number of coliforms and of Escherichia coli in the duodenum were significantly reduced by .4% BPA; in the jejunum, by all levels used in the trials; and in the ileum, by .4% and .8% of buffered propionic acid. The intestinal pH was not influenced by the BPA addition. In Trial 2, the BPA at .4% fed continuously had no adverse effect on growth, feed utilization, the abdominal fat of females, or the dressing percentage of males while significantly reducing the abdominal fat for males and increasing the dressing percentage for females. Feeding .4% BPA for the last 7 days had no effect on any of these parameters. Periodic dosage with S. typhimurium had no effect on body weight, feed utilization, or abdominal fat and significantly increased the dressing percentage. There was a significant interaction between the Salmonella dosage and the time of feeding BPA on dressing percentage.(ABSTRACT TRUNCATED AT 250 WORDS)

White Leghorn hens, housed in cages were fed commercial-type corn-soy diets containing one of three levels of 3-Nitro-10 (0, .0025, and .005%) and either of two levels of xanthophyll (high and low) supplied by yellow corn. Egg yolks were collected and the yolks analyzed for color using reflectance colorimetry. The addition of 3-Nitro-10 at either level increased egg yolk dominant wavelength (DWL) and excitation purity (EP) values while decreasing luminosity (LUM) values (probability levels: .08, .10, and .003, respectively). It was concluded that the addition of 3-Nitro-10 to the diet of laying hens improved egg yolk color.

Five trials were conducted at various locations to determine the response of broiler chickens to the addition of zinc bacitracin to diets containing both salinomycin and roxarsone. Results indicate that when a combination of salinomycin and roxarsone is used as an anticoccidial, the addition of zinc bacitracin at 33 or 55 mg/kg diet will improve growth rate and feed utilization by broiler chickens. Regression of body weight and feed utilization on dietary levels of zinc bacitracin indicated that each milligram of zinc bacitracin in the diet increased the 49-day weight of broiler chickens by .92 g and reduced the feed required per gram of gain by .0012 g.

Experiments were conducted to examine the effect of dietary roxarsone (3-nitro-4-hydroxyphenylarsonic acid) on Cu utilization by the pig, chick and rat. A fortified corn-soybean meal diet was fed in each experiment. Roxarsone dramatically reduced liver Cu concentration at all levels of supplemental Cu fed. The level of roxarsone commonly fed, 50 mg/kg diet, resulted in a two- to fourfold depression in liver Cu concentration in all species studied. The effects of roxarsone on weight gain were more perplexing. In the chick, the diets containing 100 and 250 mg Cu/kg depressed growth in the presence, but not in the absence, of 50 mg/kg dietary roxarsone. In contrast, at toxic levels of Cu, roxarsone had no effect on (500 or 750 mg Cu/kg diet) or slightly alleviated (1,000 mg Cu/kg diet) the growth-depressing effects of Cu. In the rat, a roxarsone level of 50 mg/kg diet exacerbated the growth-depressing effect of 1,000 mg Cu/kg diet. However, Cu had no effect on the growth depression that resulted from feeding a toxic level of roxarsone (250 mg/kg diet). The antagonizing effects of roxarsone and Cu on weight gain were not evident in the pig. Supplemental Cu (250 mg/kg diet) improved weight gain, but not feed efficiency, in starter pigs. Roxarsone (50 mg/kg diet) had no effect on the growth-promoting effects of Cu.

1. The response of growing male guinea fowls to dietary supplements of oxytetracycline, Roxarsone and zinc bacitracin were investigated. 2. The antibiotics produced significant increases in body weight, efficiency of food utilisation and nitrogen retention in guinea fowls at 12 weeks of age. 3. Carcase protein was not affected by antibiotic supplementation but carcase dry matter and fat were significantly higher in guinea fowls receiving medicated food than in controls. 4. The results show that the growth rate of guinea fowls is significantly increased by similar inclusion rates of oxytetracycline, Roxarsone and zinc bacitracin as those generally recommended for broiler chickens.

Three experiments were conducted to investigate the roxarsone (3-nitro-4-hydroxyphenylarsonic acid) X copper (Cu) interaction in weanling pigs. Supplemental roxarsone at 400 mg/kg diet decreased rate and efficiency of weight gain and caused visible neurological signs of toxicosis. Copper addition (CuSO4 X 5H2O) at a level of 650 mg Cu/kg diet likewise decreased weight gain and feed efficiency, and it also increased hepatic Cu deposition. The combination of these growth-depressing dosages of roxarsone and Cu resulted in a far greater reduction in gain and efficiency of feed utilization than was the case when either compound was fed alone. A growth-promoting dosage of Cu (250 mg/kg) increased weight gain by 32% in one experiment but showed no efficacy in alleviating the growth-depression resulting from feeding 400 mg/kg roxarsone. A roxarsone dosage of 100 mg/kg increased gain and feed efficiency. Surprisingly, the decreased weight gain in pigs fed 650 mg/kg Cu was ameliorated by feeding 100 mg/kg roxarsone concomitantly. This level of roxarsone also reduced liver Cu concentration substantially. It thus appears that the nature of the roxarsone X Cu interaction is dependent on the dose of each compound administered. Moreover, low-dose roxarsone administration appears to ameliorate Cu toxicity, but low-dose Cu feeding does not show efficacy against roxarsone toxicity.

Interactions among roxarsone, copper, and coccidiosis were studied in growing crossbred chicks. Corn-soy or corn-soy-corn gluten meal diets were fed in all assays. In the absence of supplemental copper, 50 mg/kg roxarsone did not affect gain. However, in the presence of 250 mg/kg supplemental copper, there was a depression in gain due to feeding 50 mg/kg roxarsone. In contrast, at a toxic level of copper (1000 mg/kg), a growth response resulted from feeding roxarsone. In all instances, roxarsone markedly decreased liver copper concentration in birds fed high levels of copper. Multiple crop intubations of Eimeria acervulina or Eimeria tenella depressed performance and exacerbated copper toxicity symptoms. Copper supplementation as well as coccidial infection resulted in depressed plasma pigmentation.

Six trials were conducted at different locations to examine the response of broiler chickens to roxarsone and bacitracin in the form of bacitracin methylene disalicylate (BMD) in the presence of the anticoccidal narasin. All diets contained narasin (80 mg/kg) and were fortified with a factorial arrangement of roxarsone (0 to 50 mg/kg) and BMD (0 or 55 mg/kg). Broilers were grown to market weights with narasin and roxarsone removed from the diets for the final 5 days. Addition of both roxarsone and BMD resulted in significant (P less than .05) improvements in body weight and feed utilization. Response to roxarsone for feed utilization was influenced to some extent by the presence or absence of BMD. Although the response to roxarsone was always positive, the degree of response was lessened by the presence of BMD. This resulted in a significant (P less than .10) interaction between roxarsone and BMD for feed utilization but not for body weight.

Bambermycins and bambermycins plus roxarsone were fed to broilers in ten consecutive experiments to determine if response (measured by body weight and feed efficiency) was continuous over a long term. Bambermycins was added at 1.1 ppm (1 to 44 days) and 2.2 ppm (45 to 51 days) to both test rations. In addition, one test ration contained roxarsone (3-nitro-4-hydroxyphenylarsonic acid) at 50 ppm (1 to 44 days). Each experiment was conducted with 1,080 commercial broiler cross-run chickens. Broilers fed bambermycins and bambermycins plus roxarsone were heavier (P less than .05) than controls in every experiment. Bambermycins plus roxarsone treated broilers were heavier (P less than .05) than bambermycins treated broilers in 2 out of 10 experiments. Broilers fed bambermycins converted feed more efficiently (P less than .05) than controls in 6 of 10 experiments and were more efficient (P less than .05) than bambermycins plus roxarsone fed broilers in 1 of 10 experiments. Bambermycins plus roxarsone treated broilers were more efficient (P less than .05) than controls in 5 of 10 experiments. These findings indicate that over a long term bambermycins and bambermycins plus roxarsone consistently and significantly (P less than .05) increased broiler body weights over controls, although the addition of roxarsone was not always effective in increasing body weights or improving feed efficiency over bambermycins fed alone.

Diammonium citrate (DAC) and urea were not used beneficially as nitrogen sources in the diet of young chicks. Supplementation of 0.43% urea to a low-protein practical diet did not significantly affect gain or feed efficiency. Adding 2.85% or 5.70% DAC to a low-protein basal depressed growth and feed efficiency significantly. Percentages of nitrogen intake absorbed and retained by chicks were depressed by DAC. When diets were balanced by added amino acids. DAC did not depress weight gains, but nitrogen percentage retention was still decreased by DAC.

The interaction between 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) and Cu was studied in a series of experiments with crossbred, broiler-type chicks. A fully fortified corn-soybean meal diet was fed in all assays. While roxarsone caused a marked reduction in liver Cu concentration, arsanilic acid (4-aminophenylarsonic acid), As2O3 and As2O5 were without effect. When structural analogs of roxarsone were studied, it was found that o-nitrophenol and 3-nitro-4-hydroxybenzoic acid also had no effect on liver Cu concentration in birds fed a high level of Cu. However, liver Co concentration was reduced by the addition of either o-nitrophenol or roxarsone to the diets of birds fed a high level of Co. It was concluded that arsenic per se had no effect on liver Cu accumulation or depletion, but that a chelate was probably formed between Cu or Co and the nitroso and hydroxyl groups of the ring portion of roxarsone. In addition to the reduction in liver Cu deposition, concentrations of Cu in the bile, brain, heart and pancreas of chicks were reduced by the addition of roxarsone to a high-Cu diet. Neither dietary nor intraperitoneally (ip) injected roxarsone had an effect on liver Cu concentration when Cu was injected ip. Therefore, both roxarsone and Cu had to be present in the diet for the liver Cu-lowering effect of roxarsone to be exerted. A further experiment was conducted with growing rats to determine the effect of roxarsone on Cu balance. Feeding roxarsone elevated Cu excretion in the urine but had no effect on Cu excretion in the feces.

Roxarsone (0.005%) medicated chickens inoculated at seven weeks of age with Eimeria maxima oocysts had significantly higher pigment levels in blood plasma than did unmedicated controls. Unmedicated E. maxima infected birds had significantly lower pigment levels compared to uninoculated controls. Uninoculated roxarsone medicated controls showed no increase in pigment levels over uninoculated unmedicated birds. This experimental design demonstrates that the beneficial effect of roxarsone medication is due to anticoccidial action rather than an increase in pigmentation after feeding the yellow-colored roxarsone. Since uninoculated birds gained more rapidly if roxarsone (0.025%) was added to the diet, the drug also appears to stimulate growth as well as to protect against coccidial infection. In two floor-pen experiments roxarsone demonstrated anticoccidial activity as measured by higher pigment scores than unmedicated infected controls. The coccidial infection was induced after seeding the litter with oocysts from birds infected with E. acervulina, E. brunetti, E. hagani, E. maxima, E. mivati, E. necatrix, E. praecox, and E. tenella. Pigmentation levels were significantly higher in plasma and skin of roxarsone medicated chickens in two experiments and in shanks in one experiment. Similar protection against depigmentation was demonstrated by adding roxarsone to feed medicated with nequinate. These results involving addition of roxarsone may explain some equivocal reports on pigmentation, weight gains or feed conversion. Some previous investigators appear to have used an inadequate experimental design by omitting use of unmedicated uninoculated controls or to have made no attempt to monitor for presence or absence of coccidiosis.