[Research Process on the Combined Pollution of Microplastics and Typical Pollutants in Agricultural Soils].

As a new type of environmental persistent pollutant， microplastics can not only have adverse effects on the ecosystem but also form complex pollution with co-existing pollutants in the surrounding environment， resulting in higher ecological and health risks. Based on the perspective of agroecosystems， this study focused on the combined pollution of heavy metals， pesticides， and antibiotics， which are three typical pollutants of farmland soil， as well as microplastics and discussed the adsorption-desorption behavior of heavy metals， pesticides， and antibiotics on microplastics. The influence of the structure and properties of microplastics， the physicochemical properties of pollutants， and environmental conditions on the adsorption and desorption behavior of heavy metals， pesticides， and antibiotics on microplastics was discussed. The influence of microplastics on the bioavailability of heavy metals， pesticides， and antibiotics in farmland soil and the internal mechanism were expounded. The existing problems and shortcomings of current research were pointed out， and the future research direction was proposed. This study can provide a scientific reference for ecological risk assessment of the combined pollution of microplastics and typical pollutants in farmland soil.

Characteristics of biological manganese oxides produced by manganese-oxidizing bacteria H38 and its removal mechanism of oxytetracycline.

Oxytetracycline (OTC) is widely used in clinical medicine and animal husbandry. Residual OTC can affect the normal life activities of microorganisms, animals, and plants and affect human health. Microbial remediation has become a research hotspot in the environmental field. Manganese oxidizing bacteria (MnOB) exist in nature, and the biological manganese oxides (BMO) produced by them have the characteristics of high efficiency, low cost, and environmental friendliness. However, the effect and mechanism of BMO in removing OTC are still unclear. In this study, Bacillus thuringiensis strain H38 of MnOB was obtained, and the conditions for its BMO production were optimized. The optimal conditions were determined as follows: optimal temperature = 35 °C, optimal pH = 7.5, optimal Mn(Ⅱ) initial concentration = 10 mmol/L. The results show that BMO are irregular or massive, mainly containing MnCO3, Mn2O3, and MnO2, with rich functional groups and chemical bonds. They have the characteristics of small particle size and large specific surface area. OTC (2.5 mg/L) was removed when the BMO dosage was 75 µmol/L and the solution pH was 5.0. The removal ratio was close to 100 % after 12 h of culture at 35 °C and 150 r/min. BMO can adsorb and catalyze the oxidation of OTC and can produce ·O2-, ·OH, 1O2, and Mn(Ⅲ) intermediate. Fifteen products and degradation pathways were identified, and the toxicity of most intermediates is reduced compared to OTC. The removal mechanism was preliminarily clarified. The results of this study are convenient for the practical application of BMO in OTC pollution in water and for solving the harm caused by antibiotic pollution.

Rhizosphere bacteria G-H27 significantly promoted the degradation of chlorpyrifos and fosthiazate.

Microbial remediation of polluted environments is the most promising and significant research direction in the field of bioremediation. In this study, chlorpyrifos and fosthiazate were selected as representative organophosphorus pesticides, wheat was the tested plant, and fluorescently labeled degrading Bacillus cereus G-H27 were the film-forming bacteria. Exogenous strengthening technology was used to establish degrading bacterial biofilms on the root surface of wheat. The influence of root surface-degrading bacterial biofilms on the enrichment of chlorpyrifos and fosthiazate in wheat was comprehensively evaluated. First, the fluorescently-labeled degrading bacteria G-H27 was constructed, and its film-forming ability was investigated. Second, the growth- promoting characteristics and degradation ability of the bacteria G-H27 were investigated. Finally, the degradation effect of the root surface-degrading bacterial biofilm on chlorpyrifos and fosthiazate was determined. The above research provides an important material basis and method for the bioremediation of pesticide-contaminated soil.

Biodegradation mechanism of chlorpyrifos by Bacillus sp. H27: Degradation enzymes, products, pathways and whole genome sequencing analysis.

Chlorpyrifos (CP) is a pesticide widely used in agricultural production. However, excessive use of CP is risky for human health and the ecological environment. Microbial remediation has become a research hotspot of environmental pollution control. In this study, the effective CP-degrading strain H27 (Bacillus cereus) was screened from farmland soil, and the degradation ratio was more than 80%. Then, the degradation mechanism was discussed in terms of enzymes, pathways, products and genes, and the mechanism was improved in terms of cell motility, secretory transport system and biofilm formation. The key CP-degrading enzymes were mainly intracellular enzymes (IE), and the degradation ratio reached 49.6% within 30 min. The optimal pH for IE was 7.0, and the optimal temperature was 25 °C. Using DFT and HPLC‒MS analysis, it was found that degradation mainly involved oxidation, hydrolysis and other reactions, and 3 degradation pathways and 14 products were identified, among which TCP (3,5,6-trichloro-2-pyridinol) was the main primary degradation product in addition to small molecules such as CO2 and H2O. Finally, the whole genome of strain H27 was sequenced, and the related degrading genes and enzymes were investigated to improve the metabolic pathways. Strain H27 had perfect genes related to flagellar assembly and chemotaxis and tended to tolerate CP. Moreover, it can secrete esterase, phosphatase and other substances, which can form biofilms and degrade CP in the environment. In addition, CP enters the cell under the action of permeases or transporters, and it is metabolized by IE. The degradation mechanism of CP by strain H27 is speculated in this study, which provided a theoretical basis for enriching CP-degrading bacteria resources, improving degradation metabolic pathways and mechanisms, and applying strain H27 to environmental pollution remediation.

Effects of magnesium-modified biochar on antibiotic resistance genes and microbial communities in chicken manure composting.

Abatement of antibiotic resistance genes (ARGs) in livestock manure by composting has attracted attention. This study investigated the effect of adding magnesium-modified biochar (MBC) on ARGs and microbial communities in chicken manure composting. Twelve genes for tetracyclines, sulfonamides, and macrolides, and mobile genetic elements were measured in the compost pile. The results showed that after 45 days of the composting, the treatment groups of MBC had longer high temperature periods, significantly higher germination indices (GI) and lower phytotoxicity. There were four major dominant phyla (Firmicutes, Actinobacteriota, Proteobacteria, and Bacteroidota) in the compost. The abundance of Firmicutes decreased significantly during the compost cooling period; tetracycline resistance genes demonstrated an extremely significant positive correlation with Firmicutes, showing a trend of the same increase and decrease with composting time; tetT, tetO, tetM, tetW, ermB, and intI2 were reduced in the MBC group; the total abundance of resistance genes in the 2% MBC addition group was 0.67 times that of the control; Proteobacteria and Chloroflexi were also significantly lower than the other treatment groups. Most ARGs were significantly associated with mobile genetic elements (MGEs); MBC can reduce the spread and diffusion of ARGs by reducing the abundance of MGEs and inhibiting horizontal gene transfer (HGT).

Immobilization of Bacillus Thuringiensis and applicability in removal of sulfamethazine from soil.

Microbial degradation is considered an essential and promising treatment for sulfadimidine contamination of soil. To address the low colonization rates and inefficiencies of typical antibiotic-degrading bacteria, sulfamethazine (SM2)-degrading strain H38 is converted into immobilized bacteria in this study. Results show that the removal rate of SM2 by immobilized strain H38 reaches 98% at 36 h, whereas the removal rate of SM2 by free bacteria reaches 75.2% at 60 h. In addition, the immobilized bacteria H38 exhibits tolerance to a wide range of pH (5-9) and temperature (20 °C-40 °C). As the amount of inoculation increases and the initial concentration of SM2 decreases, the removal rate of SM2 by the immobilized strain H38 increases gradually. Laboratory soil remediation tests show that the immobilized strain H38 can remove 90.0% of SM2 from the soil on the 12th day, which exceeds the removal by free bacteria by 23.9% in the same period. Additionally, the results show that the immobilized strain H38 enhances the overall activity of microorganisms in SM2-contaminated soil. Compared with the SM2 only (control group containing no bacteria) and free bacterial treatment groups, the gene expression levels of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, cbbLG, and cbbM increased significantly in the treatment group with immobilized strain H38. This study shows that immobilized strain H38 can reduce the effect of SM2 on soil ecology to a greater extent than free bacteria, while providing safe and effective remediation.

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg ± 1.45 mg/kg and 1.30 mg/kg ± 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg ± 0.62 mg/kg and 8.71 mg/kg ± 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

New insights into the effects of antibiotics and copper on microbial community diversity and carbon source utilization.

Residual antibiotics (ABs) and heavy metals (HMs) are continuously released from soil, reflecting their intensive use and contamination of water and soil, posing an environmental problem of great concern. Relatively few studies exist of the functional diversity of soil microorganisms under the combined action of ABs and HMs. To address this deficiency, BIOLOG ECO microplates and the Integrated Biological Responses version 2 (IBRv2) method were used to comprehensively explore the effects of single and combined actions of copper (Cu) and enrofloxacin (ENR), oxytetracycline (OTC), and sulfadimidine (SM2) on the soil microbial community. The results showed that the high concentration (0.80 mmol/kg) compound group had a significant effect on average well color development (AWCD) and OTC showed a dose-response relationship. The results of IBRv2 analysis showed that the single treatment group of ENR or SM2 had a significant effect on soil microbial communities, and the IBRv2 of E1 was 5.432. Microbes under ENR, SM2, and Cu stress had more types of available carbon sources, and all treatment groups were significantly more enriched with microorganisms having D-mannitol and L-asparagine as carbon sources. This study confirms that the combined effects of ABs and HMs can inhibit or promote the function of soil microbial communities. In addition, this paper will provide new insights into IBRv2 as an effective method to evaluate the impacts of contaminants on soil health.

Spread and driving factors of antibiotic resistance genes in soil-plant system in long-term manured greenhouse under lead (Pb) stress.

Livestock manure is often used as fertilizer in greenhouses, resulting in simultaneous enrichment of heavy metals and antibiotic resistance genes (ARGs) in soils. The soil-plant system is a non-negligible way to spread ARGs; however, the effects of lead (Pb) on the spread of ARGs and their driving factors in the greenhouse soil-plant system remain unclear. In this present study, the occurrence of ARGs in greenhouse soils and their spread into plants under Pb stress were studied. Overall, Pb promoted the accumulation of ARGs in root endophytes at 10, 50, and 100 mg/kg as well as in soils at 10 and 200 mg/kg, but reduced the total relative abundance of ARGs in leaf endophytes. Particularly, Pb increased the mobile genetic elements (MGEs) relative abundance and endophytic bacterial community diversity in roots, consistent with the change in the total relative abundance of ARGs. Network analysis revealed that bacterial community and MGEs may jointly affect the migration of ARGs in the soil-plant system of greenhouses. Overall, this study extended our knowledge of how Pb can promote the transmission of ARGs to plant roots from greenhouse soils receiving long-term manure applications, which must be considered when assessing the risk of ARGs to public health.

Effect of sulfamethazine on the horizontal transfer of plasmid-mediated antibiotic resistance genes and its mechanism of action.

As a new type of environmental pollutant, antibiotic resistance genes (ARGs) pose a huge challenge to global health. Horizontal gene transfer (HGT) represents an important route for the spread of ARGs. The widespread use of sulfamethazine (SM2) as a broad-spectrum bacteriostatic agent leads to high residual levels in the environment, thereby increasing the spread of ARGs. Therefore, we chose to study the effect of SM2 on the HGT of ARGs mediated by plasmid RP4 from Escherichia coli (E. coli) HB101 to E. coli NK5449 as well as its mechanism of action. The results showed that compared with the control group, SM2 at concentrations of 10 mg/L and 200 mg/L promoted the HGT of ARGs, but transfer frequency decreased at concentrations of 100 mg/L and 500 mg/L. The transfer frequency at 200 mg/L was 3.04 × 10-5, which was 1.34-fold of the control group. The mechanism of SM2 improving conjugation transfer is via enhancement of the mRNA expression of conjugation genes (trbBP, trfAP) and oxidative stress genes, inhibition of the mRNA expression of vertical transfer genes, up regulation of the outer membrane protein genes (ompC, ompA), promotion of the formation of cell pores, and improvement of the permeability of cell membrane to promote the conjugation transfer of plasmid RP4. The results of this study provide theoretical support for studying the spread of ARGs in the environment.

Depleting hsa_circ_0000567 suppresses acquired gefitinib resistance and proliferation of lung adenocarcinoma cells through regulating the miR-377-3p / ZFX axis: an in vitro and in vivo study.

BACKGROUND: Circular RNAs (circRNAs) expression profile has been reported in lung adenocarcinoma (LUAD) cells resistant to gefitinib, and hsa_circ_0000567 was abnormally upregulated. However, its precise role in gefitinib resistance remains unclarified. METHODS: Levels of hsa_circ_0000567, microRNA (miR)-377-3p and zinc finger protein X-linked (ZFX) were detected by real-time quantitative PCR. Direct attachment was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Gefitinib resistance was measured by MTT assay, colony formation assay, flow cytometry, western blotting, and xenograft in mice. RESULTS: Expression of hsa_circ_0000567 was upreguated in human gefitinib-resistant human LUAD tissues and cells (HCC827/GR and PC9/GR). Clinically, higher hsa_circ_0000567 correlated with advanced tumor burden. Blockage of hsa_circ_0000567 suppressed cell viability, half maximal inhibitory concentration (IC50) value, colony formation ability, and expression of Bcl-2 and proliferating cell nuclear antigen (PCNA) in HCC827/GR and PC9/GR cells under gefitinib treatment or not, accompanied with enhanced apoptosis rate and Bax expression. In vivo, tumor growth of PC9/GR cells untreated and treated with gefitinib was restrained by silencing hsa_circ_0000567. miR-377-3p was directly regulated by hsa_circ_0000567, and then targeted ZFX. Similar to hsa_circ_0000567 knockdown, overexpressing miR-377-3p inhibited chemoresistance in genitinib-resistant LUAD cells in vitro, whereas, depleting miR-377-3p was able to promote gefitinib resistance in spite of hsa_circ_0000567 knockdown. Moreover, restoring ZFX abrogated miR-377-3p-mediated chemosensitivity in genitinib-resistant LUAD cells in vitro. CONCLUSION: The hsa_circ_0000567/miR-377-3p/ZFX axis might contribute to acquired gefitinib resistance in LUAD cells both in vitro and in vivo, suggesting hsa_circ_0000567 as a novel therapeutic target in treatment of gefitinib-resistant LUAD.

Distribution of quinolone and macrolide resistance genes and their co-occurrence with heavy metal resistance genes in vegetable soils with long-term application of manure.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) has become an increasingly serious global public health issue. This study investigated the distribution characteristics and influencing factors of ARB and ARGs in greenhouse vegetable soils with long-term application of manure. Five typical ARGs, four heavy metal resistance genes (MRGs), and two mobile genetic elements (MGEs) were quantified by real-time quantitative polymerase chain reaction (qPCR). The amount of ARB in manure-improved soil greatly exceeded that in control soil, and the bacterial resistance rate decreased significantly with increases in antibiotic concentrations. In addition, the resistance rate of ARB to enrofloxacin (ENR) was lower than that of tylosin (TYL). Real-time qPCR results showed that long-term application of manure enhanced the relative abundance of ARGs in vegetable soils, and the content and proportion of quinolone resistance genes were higher than those of macrolide resistance genes. Redundancy analysis (RDA) showed that qepA and qnrS significantly correlated with total and available amounts of Cu and Zn, highlighting that certain heavy metals can influence persistence of ARGs. Integrase gene intI1 correlated significantly with the relative abundance of qepA, qnrS, and ermF, suggesting that intI1 played an important role in the horizontal transfer of ARGs. Furthermore, there was a weakly but not significantly positive correlation between specific detected MRGs and ARGs and MGEs. The results of this study enhance understanding the potential for increasing ARGs in manure-applied soil, assessing ecological risk and reducing the spread of ARGs.

Soil types influence the characteristic of antibiotic resistance genes in greenhouse soil with long-term manure application.

Composted livestock and poultry manure, which may contain antibiotic resistance genes (ARGs), is widely used as natural fertilizer in China. But the influence of soil types on ARGs is not well characterized, particularly at greenhouse sites with long-term manure application. We investigated the distribution of ARGs in the cinnamon, fluvo-aquic and saline-alkali soils in greenhouse of Yellow River Delta region, China. A total of 193 ARGs subtypes were detected, with multidrug and aminoglycoside resistance genes as the most universal ARGs subtypes. Soil types influenced the ARGs distribution, where higher levels of diversity and relative abundance of ARGs in the fluvo-aquic and saline-alkali soils compared with those in the cinnamon soils. Among abiotic factors, sand, pH and Zn contributed more to the pattern of ARGs in the cinnamon soils, whereas sand and Cd, clay and Pb contributed the most in the fluvo-aquic and saline-alkali soils respectively. Furthermore, positive correlations between the relative abundances of ARGs and mobile genetic elements (MGEs) in the fluvo-aquic soils, suggesting higher dissemination potential of ARGs in this type of soil. Overall, MGEs played a positive primary role in the ARGs distribution in greenhouse soil than heavy metal co-selection and soil physicochemical properties.

Distribution characteristics of antibiotic resistant bacteria and genes in fresh and composted manures of livestock farms.

Livestock manure is a major reservoir of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study investigated the distribution characteristics of ARB, ARGs in fresh and composted manures of traditional breading industry in rural areas in China. Samples collected were naturally piled without professional composting, and will be applied to farmland. The real-time quantitative polymerase chain reaction (qPCR) results showed the presence of ten target ARGs and two mobile genetic elements (MGEs) in the tested manure samples. The relative abundance of tetracycline and sulfonamide resistance genes (TRGs and SRGs) was generally higher than that of macrolide resistance genes (MRGs), followed by quinolone resistance genes (QRGs). There were significant positive correlations between the abundance of sul1, sul2, tetW and MGEs (intl1, intl2). In addition, the distribution of target ARGs was associated with the residual concentrations of doxycycline (DOX), sulfamethazine (SM2), enrofloxacin (ENR) and tylosin (TYL). Overall, a total of 24 bacterial genera were identified. The resistance rates of ARB were 17.79%-83.70% for SM2, followed 0.40%-63.77% for TYL, 0.36%-43.90% for DOX and 0.00%-13.36% for ENR, which showed a significant dose-effect. This study also demonstrated that the abundance of clinically relevant ARB and ARGs in chicken, swine and cow fresh manures significantly greater than that in composted manures, and chicken and swine manures had higher proportion of ARB and higher abundance of ARGs than that in cow manures.

Separate and joint eco-toxicological effects of sulfadimidine and copper on soil microbial biomasses and ammoxidation microorganisms abundances.

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.

Toxicity of enrofloxacin and cadmium alone and in combination to enzymatic activities and microbial community structure in soil.

Antibiotics and heavy metals have long-term potential toxicity to the environment, and their residuals in agricultural soils are receiving more and more attention. To evaluate the ecotoxicological effects of enrofloxacin and cadmium on soil enzymatic activities and microbial community structure, soil samples were exposed to individual and combined contaminants over 28 days. The results indicated that the toxic effects of enrofloxacin alone on soil enzymatic activities were relatively small and showed no concentration dependence. In contrast, significant inhibition of soil enzymatic activities was observed upon cadmium contamination by itself. Overall, the combination of two contaminants also has toxic effect on enzymatic activities; an antagonism between enrofloxacin and cadmium was observed. On 14 and 21 days, individual enrofloxacin and cadmium reduced average well color development (AWCD), Shannon, McIntosh, Simpson indices, and substrate utilization, except for Shannon, McIntosh, Simpson indices of the cadmium 0.4 mmol/kg treatment were higher than the control on 21 days. In general, combined treatments led to higher value of these microbial diversity indicators than those found under separate contamination, although there were some exceptions. With the increase in enrofloxacin concentration, the utilization of any carbon source by the microorganisms gradually decreased. In addition, the AWCD value and substrate utilization decreased as time increased. In the separate and combined contaminant treatments, the order of substrate utilization by soil microorganisms was aliphatics > amino acids > saccharides > metabolites. Thus, enrofloxacin and cadmium had a variable but generally negative influence on soil enzymatic activities and microbial community structure.

Individual and combined effects of enrofloxacin and cadmium on soil microbial biomass and the ammonia-oxidizing functional gene.

The negative effects of residues from antibiotics and heavy metals in agricultural soils are becoming an increasingly frequent concern. To evaluate the toxicity and interaction of antibiotics and heavy metals, enrofloxacin (ENR) and cadmium (Cd) were used as targets to study the individual effects of ENR (0.025, 0.1, 0.4mmol/kg) and Cd (0.4mmol/kg) and their combined effects (mole ratios of ENR to Cd of 1: 1, 1: 4 and 1: 16) on soil microbial biomass and function on days 7, 14, 21 and 28 of the study. The results demonstrated that microbial populations, which were counted during 4 sampling periods, were mainly in the order of bacteria>actinomycetes>fungi. The ammonia monooxygenase (amoA) gene copies of ammonia-oxidizing archaea (AOA) were more abundant than ammonia-oxidizing bacteria (AOB) on days 14 and 21. Soil bacteria, fungi, and actinomycetes numbers and amoA gene abundances of AOB and AOA in soils were inhibited to varying degrees by the single and combined effects of ENR and Cd; the higher the concentration of the treatments, the stronger the inhibition. The combined toxicity of ENR and Cd on soil microbes and AOA- and AOB-amoA genes was stronger than when either chemical was used alone; the interaction effects of ENR and Cd were mainly antagonistic. Moreover, the ratios of bacteria/fungi declined significantly on days 14, 21 and 28; the proportions of AOA- and AOB-amoA were altered with the addition of ENR and Cd. Thus, ENR and Cd had significant negative effects on the soil microbial community, especially when both contaminants were present.

Toxic effects of oxytetracycline and copper, separately or combined, on soil microbial biomasses.

The production of commercial livestock and poultry often involves using with antibiotics and feed additives, such as oxytetracycline (OTC) and copper (Cu). These are often excreted into the soil by animal feces; hence, combined pollutants may contaminate the soil. To evaluate single and combined toxic effects of OTC and Cu on the soil ecology, changes in quantities of bacteria, fungi, and actinomycetes in the soil were studied over a 28-d incubation period by a plate count method, microbes numbers counted on days 7, 14, 21, and 28. Abundances of ammonia monooxygenase (amoA) gene expression by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in soil samples also were tested by real-time polymerase chain reactions (RT-PCRs) on day 21. The results revealed that the numbers of bacteria, fungi and actinomycetes and amoA genes copies of AOA and AOB were reduced seriously by exposure to Cu (1.60 mmol/kg). Similarly, the combined pollution treatments (mole ratios of OTC: Cu was 1:2, 1:8, and 1:32) also had inhibitory effect on bacteria, fungi, and actinomycetes numbers and amoA gene copies of AOA and AOB; the inhibitory rate was on obvious growth trend with the increasing mole ratios. Effects from single OTC pollution were found on bacteria (days 7 and 14), fungi (days 7, 14, 21, and 28), and AOA-amoA gene copies (day 21), with promotion at a low concentration (0.05 mmol/kg) and suppression at higher concentrations (0.2 and 0.8 mmol/kg). Also, numbers of bacteria, fungi, and actinomycetes decreased with longer culture times. Combining OTC and Cu led to a higher inhibition of soil microbes than when either chemical was used alone. However, there was no significant relationship between single and combined toxic chemicals because of their complicated interactions, either antagonistic or synergistic. The results also indicated the sensitivity of bacteria, fungi, actinomycetes on toxic chemicals existed difference and that the AOA were more tolerant than the AOB to these chemicals.

[Influence of Four Kinds of PPCPs on Micronucleus Rate of the Root-Tip Cells of Vicia-faba and Garlic].

In order to determine the degree of biological genetic injury induced by PPCPs, the genotoxic effects of the doxycycline (DOX), ciprofloxacin (CIP), triclocarban (TCC) and carbamazepine (CBZ) in the concentration range of 12.5-100 mg · L⁻¹ were studied using micronucleus rate and micronucleus index of Vicia-fabe and garlic. The results showed that: (1) When the Vicia-faba root- tip cells were exposed to DOX, CIP, TCC and CBZ, micronucleus rates were higher than 1.67 ‰ (CK1), it was significantly different from that of the control group (P < 0.05), and the micronucleus index was even greater than 3.5; With the increasing concentrations of the PPCPs, the micronucleus rates first increased and then decreased. (2) When the garlic root tip cells were exposed to DOX, CIP, TCC and CBZ respectively, the micronucleus rates were less than those of the Vicia-faba, while in most treatments significantly higher than that of the control group (0.67‰). The micronucleus index was higher than 3.5 in the groups exposed to CIP with concentrations of 25, 50, 100 mg · L⁻¹ and TCC and CBZ with concentrations of 25 mg · L⁻¹; With the increase of exposure concentrations, the micronucleus rate showed a trend of first increasing and then decreasing as well. (3) Under the same experimental conditions, the cells micronucleus rates of the garlic cells caused by the four tested compounds were significantly lower than those of Vicia-faba. (4) The micronucleus index of the root tip cells of Vicia-faba and garlic treated with the four kinds of compounds followed the order of CIP > CBZ > TCC > DOX. These results demonstrated that the four compounds caused biological genetic injury to root-tip cells of Vicia-faba and garlic, and the genetic damage caused to garlic was significantly lower than that to Vicia-faba. The damages caused by the four kinds of different compounds were also different.

Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors.

Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca(2+) signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca(2+) homeostasis. To monitor intracellular Ca(2+) concentration ([Ca(2+)](i)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in [Ca(2+)](i) in a physiological Ca(2+)-containing solution, with an EC(50) of 2.5 microM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca(2+)](i), and the P1-receptor agonist adenosine caused only a small increase in [Ca(2+)](i). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca(2+)](i), indicating P2Y receptors were involved in this process. In a Ca(2+)-free bath solution, thapsigargin and ATP induced intracellular Ca(2+) release from an identical pool. Nucleotides caused an increase in [Ca(2+)](i) in the potency order of UTP = ATP > ATP gamma S > ADP > UDP that is best fitted with the P2Y(2) subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca(2+)](i) as did ATP, a small but sustained increase in [Ca(2+)](i) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in [Ca(2+)](i) could be blocked by either nonselective cation channel blockers Gd(3+) or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd(3+) or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca(2+)](i) was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X(1) and P2Y(2) receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca(2+) signaling.