Nonlinear relationship between viral load and TCT in single/multiple HPV52 infection.

PURPOSE: To determine the correlation between HPV (human papillomavirus) 52 viral load, multiple infections and ThinPrep cytology test (TCT), to inform clinical management of HPV52-positive women after cervical cancer screening. METHODS: A total of 1,882 female patients who had positive quantitative HPV tests at Yuebei People's Hospital from January 2020 to December 2022, of whom 533 tested positive for HPV52. We excluded patients who combined HPV16 and/or HPV 18 positivity and whom HPV52 viral load could not be calculated. The final enrollment was 488 patients, including 400 NILM, 48 ASC-US, 28 LSIL and 12 HSIL. The HPV test is a quantitative multiplexed fluorescent PCR assay that provides both HPV genotyping and viral load. RESULTS: In our study, there were differences in the median distribution of viral loads among various cytological class categories. The risk of TCT results (LSIL or worse) was increased with the increase of HPV52 viral load, for every LOG unit increase in HPV52 viral load, the risk increased by 26.6%. More importantly, we found a nonlinear relationship between HPV52 viral load and TCT results (LSIL or worse) in both single and multiple infections. When the viral load reaches a threshold, the risk of abnormal cytological results increases significantly. CONCLUSION: HPV52 viral load is an independent risk factor for TCT results (LSIL or worse). The relationship between HPV52 viral load and TCT results (LSIL or worse) is not linear. Viral load may be used as a triage indicator for HPV52-positive patients, thus improving the post-screening clinical management of HPV52-positive women.

Improved 2α-Hydroxylation Efficiency of Steroids by CYP154C2 Using Structure-Guided Rational Design.

Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C-H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs.

The 16α-Hydroxylation of Progesterone by Cytochrome P450 107X1 from Streptomyces avermitilis.

Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous heme-dependent enzymes that catalyze the monooxygenation of non-activated C-H bonds to modify the structure of the substrate. In this study, we heterologously expressed CYP107X1 from Streptomyces avermitilis and conducted in vitro substrate screening using the alternative redox partners putidaredoxin and putidaredoxin reductase. CYP107X1 catalyzed the 16α-hydroxylation of progesterone with regio- and stereoselectivity. The spectroscopic analyses showed that CYP107X1 bound progesterone with a relatively high Kd value of 65.3±38.9 µM. The Km and kcat values for progesterone were estimated to be 47.7±12.0 µM and 0.30 min-1 , respectively. Furthermore, a crystal structure was obtained of CYP107X1 bound with glycerol from the buffer solution. Interestingly, a conserved threonine was replaced with asparagine in CYP107X1, indicating that it may adopt an unnatural proton transfer process and play a crucial role in its catalytic activity.

Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7.

Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C-H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate-promiscuous P450 which catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as Km =311.60±87.30 µM and kcat =2.01±0.33 min-1 . In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.

Dietary taurine supplementation ameliorates muscle loss in chronic heat stressed broilers via suppressing the perk signaling and reversing endoplasmic reticulum-stress-induced apoptosis.

BACKGROUND: Heat stress seriously affects animal health and induces enormous financial losses in poultry production. Exploring the appropriate means for ameliorating unfavorable effects caused by heat stress is essential. We investigated whether taurine supplementation could attenuate breast muscle loss in chronic heat-stressed broilers, as well as its mechanism. We designed three groups: a normal control group (22 °C), a heat stress group (32 °C) and a taurine treatment group (32 °C, basal diet + 5 g·kg-1 taurine). RESULTS: We found that taurine significantly moderated the decreases of breast muscle mass and yield, as well as the increases of serum aspartate aminotransferase activity and serum urine acid level in chronic heat-stressed broilers. Additionally, supplementary taurine significantly alleviated elevations of the cytoplasm Ca2+ concentration, protein expressions of GRP78 and p-PERK, mRNA expressions of Ca2+ channels (RyR1, IP3R3) and endoplasmic reticulum (ER) stress factors (GRP78, GRP94, PERK, EIF2α, ATF4, IRE1, XBP1, ATF6 and CHOP), apoptosis (Caspase-3 and TUNEL), protein catabolism, and the reduction of taurine transporter (TauT) mRNA expression in the breast muscle induced by chronic heat stress. CONCLUSION: Supplementary taurine could attenuate chronic heat stress-induced breast muscle loss via reversing ER stress-induced apoptosis and suppressing protein catabolism. © 2020 Society of Chemical Industry.

Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis.

Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C-H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners-RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The Km and kcat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min-1, and kcat/Km was 0.81 min-1 mM-1. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.

Hydroxylation of Steroids by a Microbial Substrate-Promiscuous P450 Cytochrome (CYP105D7): Key Arginine Residues for Rational Design.

Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2ß- and D-ring 16ß-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

MiR-106b promotes therapeutic antibody expression in CHO cells by targeting deubiquitinase CYLD.

MicroRNAs (miRNAs) function as important regulators of major cellular processes, such as cell cycle, proliferation, development, and apoptosis. Recently, miRNA engineering of Chinese hamster ovary (CHO) cells has emerged as a promising strategy for enhancing therapeutic antibody production. Previously, we have reported that inhibition of deubiquitinase cylindromatosis (CYLD) remarkably enhanced the therapeutic antibody production in CHO cells. However, the mechanisms regulating CYLD in CHO cells remain elusive. Herein, we demonstrated that miR-106b targets CYLD directly, as shown by a series of bioinformatics analyses and experimental assays. Stable overexpression of miR-106b in CHO cells promoted CHO cell viability and subsequent antibody expression in transient transfection assay. Furthermore, the results in fed-batch culture showed that stable overexpression of miR-106b in a CHO-IgG cell line achieved about 0.66-fold promotion in product titer compared to the parental cells. Meanwhile, overexpression of miR-106b did not affect the quality of antibody. Taken together, our findings highlight the effect of miR-106b inhibition in CYLD synthesis and its function in antibody expression as a new target for improving CHO manufacturing cells.

Chronic heat stress alters hypothalamus integrity, the serum indexes and attenuates expressions of hypothalamic appetite genes in broilers.

The hypothalamus is crucial to ensure the functionality of the entire organisms, such as body temperature, feed intake and energy regulation. Exposing broilers to high ambient temperature usually induces lower feed intake and energy imbalance. We investigated the molecular mechanisms by which heat stress impairs the appetite via dysfunction in hypothalamus of the broilers. Broilers were allocated to three groups: the normal control (NC) group, and fed ad libitum; heat-stress (HS) group, and fed ad libitum; pair-fed (PF) group, which received the feed intake equal to HS group. Experiment lasted from the age of 28 to 42 d. The results showed that HS increased the head surface temperature of broiler and changed hypothalamic ultrastructure. HS treatment also increased the serum corticosterone in the broilers after 7 days of heat stress, elevated the FT4 and FT3 after 14 days of heat stress. Heat stress of 14 days showed a tendency to increase the leptin. However, the serum corticosterone in the HS group had no significant difference after 14 days of heat stress. In addition, HS treatment decreased the expression of orexigenic gene neuropeptide Y (NPY) after 14 days of heat stress, while HS treatment had no effect on the reactive oxygen species (ROS), as well as the gene expression of AMPKα1 and LKB1 in the hypothalamus. In conclusion, HS increased the surface temperature of head in broiler, and then altered the integrity of hypothalamus. Meanwhile, HS increased the serum corticosterone which may ascribe to the activation of HPA axis in the broilers. In addition, chronic heat stress decreased the expression of orexigenic gene NPY, which may cause the broiler to reduce feed intake.

Dietary taurine supplementation improves breast meat quality in chronic heat-stressed broilers via activating the Nrf2 pathway and protecting mitochondria from oxidative attack.

BACKGROUND: Chronic heat stress can induce oxidative impairment and decrease breast meat quality in broilers. Taurine is a ß-amino acid with antioxidant properties. To investigate the alleviative effects and molecular mechanisms of taurine supplementation on breast meat quality in broilers exposed to chronic heat stress, 144 28-day-old chickens (Arbor Acres) were randomly distributed to thermoneutral (TN, 22 °C, basal diet), heat stress (HS, consistent 32 °C, basal diet), or heat stress plus taurine (HS + T, consistent 32 °C, basal diet + 5.00 g kg-1 taurine) groups for a 14-day trial. RESULTS: Chronic heat stress did not affect the contents of moisture, crude protein and crude fat in breast muscle, but impaired breast meat quality in broilers. Taurine supplementation significantly alleviated the increase in lightness and drip loss and the decrease in pH45 min and shear force of breast meat in chronic heat-stressed broilers. Compared with the HS group, taurine supplementation significantly decreased the levels of reactive oxygen species and malonaldehyde and increased the messenger RNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 and heme oxygenase 1 in the HS + T group. Meanwhile, taurine supplementation effectively alleviated mitochondrial damage caused by chronic heat exposure. CONCLUSION: Dietary taurine supplementation can effectively improve the quality of breast meat in chronic heat-stressed broilers via activating the Nrf2 pathway and protecting mitochondria from oxidative attack. © 2018 Society of Chemical Industry.

Dietary taurine supplementation decreases fat synthesis by suppressing the liver X receptor α pathway and alleviates lipid accumulation in the liver of chronic heat-stressed broilers.

BACKGROUND: Chronic heat stress can enhance fat synthesis and result in lipid accumulation in the liver of broilers. To investigate the effects and molecular mechanisms of dietary taurine supplementation on fat synthesis and lipid accumulation in the liver of chronic heat-stressed broilers, 144 28 day-old chickens (Arbor Acres) were randomly distributed to normal control (NC, 22 °C, basal diet), heat stress (HS, consistent 32 °C, basal diet), or heat stress plus taurine (HS + T, consistent 32 °C, basal diet +5.00 g kg-1 taurine) groups for a 14-day feeding trial. RESULTS: Compared with those of the HS group, dietary taurine supplementation significantly decreased the level of very-low-density lipoprotein and the activity of aspartate aminotransferase in plasma and the relative weight of liver in the HS + T group. In addition, dietary taurine supplementation also significantly decreased the levels of triglyceride, acyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and suppressed the mRNA expression levels of liver X receptor α (LXRα), sterol response element-binding protein 1c, ACC and FAS in the liver of chronic heat-stressed broilers. Meanwhile, dietary taurine supplementation effectively alleviated lipid accumulation in the liver of broilers exposed to chronic heat stress. CONCLUSION: Chronic heat stress significantly increased fat synthesis and resulted in excess lipid deposition in the liver of broilers. Dietary taurine supplementation can effectively decrease fat synthesis by suppressing the LXRα pathway and alleviate lipid accumulation in the liver of chronic heat-stressed broilers. © 2019 Society of Chemical Industry.

Contributions of Zn Ions to ZnO Nanoparticle Toxicity on Microcystis aeruginosa During Chronic Exposure.

In this work, we assessed the toxic effects of ZnO nanoparticles (NPs; 1, 10, and 50 mg L-1) and the corresponding dissoluble Zn ions (0.71, 8.66, and 35.59 mg L-1) on Microcystis aeruginosa. After chronic exposure (28 days), significantly higher growth inhibition was observed under ZnO NPs at 1 mg L-1 (47%) than under Zn ions at 0.71 mg L-1 (-15%). The opposite effect pattern was observed for ZnO NPs at 10 (71% vs. 80%) and 50 mg L-1 (73% vs. 95%) compared to Zn ions at the corresponding concentrations. After 7 days of exposure, ZnO NPs at 10 and 50 mg L-1 led to an increase of 83 and 53% in malondialdehyde content, as well as an increase of 106 and 61% in superoxide dismutase activity, respectively. However, Zn ions at the corresponding concentrations showed negligible impacts on the two parameters. The different results indicate that the insoluble NPs during the initial exposure mostly account for lipid peroxidation, which further lead to microalgal antioxidant response. During the subsequent exposure, the contributors of ZnO NP toxicity shift with the concentration and exposure time of ZnO NPs. In conclusion, the study presents new insights into the different contributions of insoluble NPs and dissoluble metallic ions to metallic NP toxicity.

Serum metabolomics study of nutrient metabolic variations in chronic heat-stressed broilers.

To investigate the effects of heat stress on broiler metabolism, we assigned 144 broilers to normal control (NC), heat stress (HS) or pair-fed (PF) groups and then monitored the effects using growth performance, carcass characteristics, biochemical assays and GC-MS-based metabolomics. The up-regulation of cloacal temperature confirmed that our experiment was successful in inducing chronic heat stress. The average daily gain and average daily feed intake of the HS group were significantly lower than those of the NC group, by 28·76 and 18·42 %, respectively (P1 and P<0·05). The greater feed:gain ratio of the HS group was significantly positively correlated with the leg, abdominal fat, subcutaneous fat and intramuscular fat proportions and levels of some free amino acids (proline, l-cysteine, methionine and threonine) but was negatively correlated with breast proportion and levels of some NEFA (stearic acid, arachidonic acid, palmitic acid and oleic acid). These findings indicated that the heat-stressed broilers were in negative energy balance and unable to effectively mobilise fat, thereby resulting in protein decomposition, which subsequently affected growth performance and carcass characteristics.

Toxicological sensitivity of Pennisetum americanum (L.) K. Schum to atrazine exposure.

The tolerance of Pennisetum americanum (L.) K Schum (P. americanum) to the herbicide atrazine, as well as the atrazine accumulation in exposed plants, was investigated in this study. The germination of P. americanum seeds was not significantly inhibited by exposure to atrazine at concentrations below 100 mg·L-1. The roots of the seedlings were much more sensitive to atrazine than the shoots were, as shown by observations that shoot and root elongation were significantly inhibited by treatment with 100 and 50 mg·kg-1 of atrazine, respectively. In addition, significant differences were found in the dry weights of the seedling shoots and roots after exposure to 50 and 20 mg·kg-1 of atrazine, respectively. Atrazine accumulated readily in the roots of exposed seedlings, and the root ultrastructure was visibly damaged by exposure to lower levels of atrazine compared to the ultrastructure of the shoot. The contents of chlorophyll a and chlorophyll b and the transcription of psbA were inhibited by exposure to atrazine at concentrations above 20 mg·kg-1. Finally, the tolerance threshold of P. americanum to atrazine was about 20 mg·kg-1, indicating that the test plant exhibited some atrazine tolerance.

Physiological and molecular responses of pearl millet seedling to atrazine stress.

Pearl millet has been recommended beneficial for several therapeutic purposes. However, little is known of the physiological responses to abiotic stressors, especially of atrazine. In order to elucidate the physiological and molecular responses of pearl millet to atrazine stress, we studied the response of various biomarkers under increasing herbicide concentrations (0, 5, 10, and 50 mg/kg). We also quantified the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (H2O2 and O2•-) produced in the leaves to evaluate the extent of oxidative damage. Increasing atrazine concentrations significantly increased ROS and MDA production in the plant leaves. Ascorbate peroxidase (APX) and peroxidase (POD) activities increased, while catalase (CAT) and superoxide dismutase activities reduced with increasing atrazine concentrations. Generally, atrazine applied at 50 mg/kg suppressed chlorophyll contents, whereas, chlorophyll (a/b) ratio was increased. Atrazine applied at 50 mg/kg significantly suppressed antioxidant gene expressions to the lowest. The APX gene showed overall low response to the atrazine treatments. The chloroplastic psbA gene showed highest expression with 10 mg/kg atrazine, whereas atrazine at 50 mg/kg significantly suppressed the gene expression to its lowest. Pearl millet was able to suppress oxidative stress under low atrazine levels, but high atrazine concentration could induce more oxidative damage.

Effects of chronic heat exposure on growth performance, intestinal epithelial histology, appetite-related hormones and genes expression in broilers.

BACKGROUND: Heat stress often occurs in the modern poultry industry, which impairs growth performance, particularly via reducing appetite. This study was aimed to investigate the mechanism of attenuating appetite by chronic heat exposure in broilers. A total of 144 broilers (28 days old) were allocated to normal control (NC, 22 °C), heat stress (32 °C), and pair-fed (22 °C) groups. RESULTS: Chronic heat exposure significantly increased cloacal temperatures and respiration rates, decreased the average daily feed intake and average daily gain, increased feed-to-gain ratio compared with the NC group, and elevated the concentrations of ghrelin and cholecystokinin (CCK) both in serum and intestine, as well as peptide YY and somatostatin in intestine on 35- or 42-day-old broilers. Moreover, heat exposure decreased villi height (VH) and the ratio of VH to crypt depth (CD), while it increased CD in the jejunum on 35- and 42 day-olds, increased (P < 0.05) the concentrations of valine and isoleucine in plasma on 42 days, and upregulated (P < 0.05) the expression of taste receptor type 1 members 1 and 3 (T1R1 and T1R3), CCK, and ghrelin in the intestine on 35- or 42-day-old broilers. CONCLUSION: Chronic heat exposure impairs the performance, intestinal morphology and appetite, which may be correlated with the increased secretion or gene expression of appetite-related hormones and genes, and the higher expression of nutrient-sensing receptors (T1R1 and T1R3) in broilers. © 2018 Society of Chemical Industry.

Determination of Pb, Cr, Cd, and As in Aluminum-Plastic Packaging Materials via Inductively Coupled Plasma-Mass Spectrometry with Microwave Digestion.

Microwave digestion was performed to study the pretreatment methods of aluminum-plastic packaging materials (APPMs). Five different digestion reagent combinations and proportions were thoroughly considered. Digestion results indicated that the most suitable reagent combination was sulfuric and nitric acids with the optimal proportion of 1∶7 after the orthogonal experiment. Moreover, the possible reasons of the experimental phenomenon were analyzed. The contents of Pb, Cr, Cd, and As in APPMs were subsequently determined via inductively coupled plasma-mass spectrometry (ICP-MS). The satisfactory linearity of calibration curves was obtained with the linear correlation coefficients above 0.999 5, and the instrument detection limits of Pb, Cr, Cd, and As for the current method were 0.215, 0.067, 0.006 and 0.020 ng·mL-1, respectively. Furthermore, the recoveries of standard addition ranged from 83.8% to 111.6%, and the relative standard deviations ranged from 0.5% to 7.4%. Two independent parallel determination results of Pb, Cr, Cd, and As in APPMs were approaching, and the student's t-test (confidence level, α=0.05) showed that the determination results had no significant differences. In conclusion, the present method exhibited fine linearity, low detection limit, high recovery, and good precision, which can accurately be utilized to analyze Pb, Cr, Cd, and As elements in APPMs or other similar materials.

Exogenous calcium induces tolerance to atrazine stress in Pennisetum seedlings and promotes photosynthetic activity, antioxidant enzymes and psbA gene transcripts.

Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented.

Enzymatic antioxidant defense in resistant plant: Pennisetum americanum (L.) K. Schum during long-term atrazine exposure.

Plants belonging to the genus Pennisetum have been reported to be resistant to atrazine, a widely used herbicide that also can cause serious pollution of soil and water. To evaluate the enzymatic antioxidant defense mechanism to the oxidative stress of atrazine, experiments focusing on the malondialdehyde (MDA) content and antioxidant enzyme in the leaf and root of Pennisetum americanum (L.) K. Schum (P. americanum) during long-term (68days) atrazine exposure were carried out. The test plant had not suffered obvious lipid membrane peroxidation, which was further confirmed by the result that the MDA content in the root and the leaf of the test plant did not significantly increase when treated with various concentrations of atrazine. The activity of the well-known antioxidases, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD), was increased when the plants were exposed to atrazine, especially at moderate concentrations (20mgkg-1 or below). These results revealed that antioxidant enzymes played important roles in protecting P. americanum from the oxidative damage induced by atrazine. The increased and more stable SOD activity in the leaf compared to in the root portion of the plant under increasing atrazine concentrations and increasing exposure time indicated that the leaf exhibited more pronounced superoxide radical scavenging ability than the root. Furthermore, correlation analysis showed that the studied antioxidases were positively correlated with the exposure time, suggesting that the antioxidant defense in P. americanum seedlings might become stronger as the plant matures. In conclusion, the increasing antioxidant enzyme activities enable P. americanum seedlings to cope with the oxidative stress induced by moderate concentrations (20mgkg-1 or below) of atrazine.

Oxidative stress response induced in an atrazine phytoremediating plant: physiological responses of Pennisetum glaucum to high atrazine concentrations.

This research presented here, for the first time, elucidates the responses of several antioxidants in Pennisetum leaves exposed to varying concentrations of atrazine (0 - 200 mg•kg-1). Pennisetum has been reported to be resistant to atrazine; however, its physiological response to high concentrations (≥ 50 mg•kg-1) of atrazine is not well documented. The contents of reduced (AsA) and oxidized (DHA) ascorbate increased significantly with increase in atrazine concentration and exposure time; but the increase was more evident under higher (50 and 100 mg•kg-1) atrazine concentrations. Increase in atrazine concentration to 200 mg•kg-1 significantly decreased AsA, but increased DHA content, throughout the experiment. Seedlings treated with 200 mg•kg-1 atrazine showed significantly lowest reduced glutathione (GSH) content; while oxidized glutathione (GSSG) was not significantly affected, after 68d. Seedlings treated with 100 mg•kg-1 atrazine showed increased Glutathione-S-Transferase (GST) activity after 48 d and 68 d; while treatment with 200 mg•kg-1 atrazine significantly increased Glutathione reductase (GR) after 58d. This result suggests that Pennisetum may tolerate lower atrazine concentrations; However, higher concentrations (≥50 mg•kg-1) which could have longer residency period in the soil, could induce more physiological damage to the plant.