Nonsteroidal anti-inflammatory drug diclofenac accelerates the emergence of antibiotic resistance via mutagenesis.

Overuse of antimicrobial agents are generally considered to be a key factor in the occurrence of antibiotic resistance bacteria (ARB). Nevertheless, it is unclear whether ARB can be induced by non-antibiotic chemicals such as nonsteroidal anti-inflammatory drug (NSAID). Thus, the objective of this study is to investigate whether NSAID diclofenac (DCF) promote the emergence of antibiotic resistance in Escherichia coli K12 MG1655. Our results suggested that DCF induced the occurrence of ARB which showed hereditary stability of resistance. Meanwhile, gene variation was identified on chromosome of the ARB, and DCF can cause bacterial oxidative stress and SOS response. Subsequently, transcriptional levels of antioxidant (soxS, sodA, sodC, gor, katG, ahpF) and SOS (recA, lexA, uvrA, uvrB, ruvA, ruvB, dinB, umuC, polB) system-related genes were enhanced. However, the expression of related genes cannot be increased in high-dosage treatment compared with low-dosage samples because of cytotoxicity and cellular damage. Simultaneously, high-dosage DCF decreased the mutation frequency but enhanced the resistance of mutants. Our findings expand our knowledge of the promoting effect on the emergence of ARB caused by DCF. More attention and regulations should be given to these potential ecological and health risks for widespread DCF.

Ephedra sinica mitigates hepatic oxidative stress and inflammation via suppressing the TLR4/MyD88/NF-κB pathway in fipronil-treated rats.

Ephedra sinica (ES) is a promising medicinal plant with a wide range of pharmacological aspects, including antioxidant and anti-inflammatory properties. Fipronil (FN) is a popularly used systemic insecticide in agriculture and veterinary applications. FN exposure can result in a variety of negative health consequences. The study aimed to explore the prophylactic effects of Ephedra sinica extract (ESE) against hepatotoxicity in FN-treated rats by following the TLR4/ MyD88/ NF-κB pathway. ESE was tested for polyphenolic and antioxidant activity. Forty rats were separated into four groups and given orally by FN (10 mg/kg B.W.) and/or ESE (150 mg/kg B.W.). Blood and tissue samples were collected at the end of the experiment and prepared for pathophysiological, gene expression, and pathological analysis. ESE showed strong antioxidant activity, as well as reduced levels of hepatic MDA and oxidative stress markers (H2O2, NO). Hepatic SOD and CAT activities were increased even further. Furthermore, in FN-treated rats, ESE improved liver functions (ALT, AST, ALP, and LDH) and recovered the lipid profile (Cho, TriG, HDL, and LDL). Moreover, by inhibiting TLR4/ MyD88/ NF-κB induction, ESE alleviated hepatic pathological changes and decreased FN-induced elevations of TNF-α, IL-6, and IL-1ß mRNA/protein levels. These findings suggested that ESE mitigated FN-induced hepatotoxicity via combating oxidative stress and relieving inflammation.

Zingiber officinale ethanolic extract attenuates oxidative stress, steroidogenic gene expression alterations, and testicular histopathology induced by sodium arsenite in male rats.

Arsenic (As) indelibly exists in the environment and may reach to a food chain. Flavors and herbs are recognized sources of natural antioxidants that play imperative against harmful chemical pollutants. Ginger is utilized around the world as a zesty condiment. This study assessed the ability of ginger extract (GE) as a protector to improve regenerative disabilities initiated by sodium arsenate in reproductive functions in male rats. Thirty-two Sprague-Dawley male rats weighted 240 ± 10 g were arbitrarily relegated into four experimental groups (n = 8): the control group; the GE-treated group received at 100 mg/kg BW; the As-treated group received sodium arsenite at 10 mg/kg BW; the fourth group received sodium arsenite additionally GE at mentioned doses for 4 weeks. Phytochemical results of GE revealed that GE had good antioxidative characteristics and high content of total flavonoid, tannins, alkaloids, and total phenolic components. Simultaneously, treatment of GE showed protection against oxidative stress induced by As and restoration of the serum cholesterol, testosterone, LH, and sperm parameter to normal levels. GE significantly improved the antioxidant activities (GSH, SOD, and CAT) as well as H2O2 and MDA in rats received concurrently the GE and As compared with control group. Moreover, the expression of genes controlling the cholesterol transportation and testosterone synthesis (SR-B1, StAR, CYP11A1, 3b-HSD, 17b-HSD, and CYP17a) as well as LHR showed a meaningful improvement in rats treated by GE plus As compared with their expression in the As-treated group. Besides, GE treatment exhibited significant recovered testis histopathological alterations, reduced the arsenic content in testes, and improved the sperm parameters.

N-acetylcysteine alleviated bisphenol A-induced testicular DNA hypermethylation of rare minnow (Gobiocypris rarus) by increasing cysteine contents.

Ubiquitous BPA exposure resulted in DNA methylation errors and oxidative stress. Numerous studies have demonstrated that oxidative stress can lead to changes in DNA methylation levels and supplementation with antioxidants, including N-acetylcysteine (NAC), was able to restore these changes. Our previous study supposed that BPA-induced de novo synthesis of glutathione (GSH) promoted DNA methylation process in Gobiocypris rarus testes. To validate this conjecture and explore the protective effects of NAC on BPA toxicity, the present study was carried out. Adult male G. rarus was treated with 225 µg L-1 BPA and/or NAC for 7 days. The sperm motility and DNA integrity of G. rarus were determined. Meanwhile, the levels of 5-methylcytosine (5mC), GSH, hydrogen peroxide (H2O2), DNA methyltransferase proteins (DNMTs), γ-glutamyl cysteine synthetase (GCS), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), nicotinamide adenine dinucleotide phosphate (NADPH) and cysteine in the testes were detected. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured. Results indicated that NAC addition resulted in increase of cysteine contents and partially inhibited the BPA-induced DNA hypermethylation of G. rarus testes. In addition, the changes in DNA methylation levels in the testes after BPA and/or NAC treatment might be controlled by DNA methylation process that mediated by DNMTs. Moreover, BPA exposure caused oxidative stress in the testes and the elimination of H2O2 might be mainly accomplished by CAT while it changed to mainly through GPx after NAC supplement. Finally, the positive response of testicular antioxidant enzyme system and the antioxidant activity of NAC itself protected sperm motility and DNA integrity from oxidative damage in each group.

Gene expression profiling of key genes in hypothalamus-pituitary-gonad axis of rare minnow Gobiocypris rarus in response to EE2.

The 17α-ethinylestradiol (EE2), which could induce estrogenic effects, is found in different aquatic systems. The current study aimed to assess in vivo effects of short-term EE2 exposure on the transcriptional activity of genes in the brain and gonad tissues in order to characterize the mode of action of EE2 on the hypothalamus-pituitary-gonad axis in rare minnow (Gobiocypris rarus). The full length cDNAs of fshß, lhß, fshr and lhr were first characterized in G. rarus. The homology and phylogenetic analyses of the amino acid sequences revealed that these four genes share high identity in cyprinid fish. The tissue distribution analysis by qRT-PCR showed that fshß and lhß were mainly expressed in the brain and fshr and lhr were mainly expressed in gonads. Adult G. rarus was exposed to EE2 at 1, 5, 25 and 125 ng/L for 3 and 6 days and the expression of brain cyp19a1b, fshß and lhß, estrogen receptors (esr1, esr2a, and esr2b) and gonadal fshr, lhr and cyp19a1a were assessed. Cyp19a1b was significantly up-regulated in the brains of female exposed to EE2 at 1-125 ng/L for 6 days. The brain lhß, but not fshß was strongly suppressed in most EE2 exposure groups of both sexes. The brain esr2b was inhibited in both sexes exposed to EE2 at all of the four concentrations for 6 days. Esr2a was up-regulated in the females by 6-day EE2 treatment at 1 and 25 ng/L. The high responsiveness of brain lhß and esr2s to EE2 and their significant correlation in both sexes suggested that the transcriptional activity of Esr2s could play key roles in modulation of lhß expression via direct action on gonadotropic cells in response to EE2. In gonads, fshr was strongly inhibited by EE2 in males, while lhr was significantly stimulated by EE2 in females. Cyp19a1a was inhibited by EE2 in both sexes. The positive correlations of gene expressions of both fshr and lhr with cyp19a1a in testes suggest that the suppression of 17α-estradiol (E2) synthesis in testis by exogenous estrogen could mediate via both Fsh/Fshr and Lh/Lhr signaling in male G. rarus.

Molecular cloning of Foxl2 gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow, Gobiocypris rarus.

Endocrine-disrupting chemicals (EDCs) can affect normal sexual differentiation in fish. Foxl2, one forkhead transcription factor, plays an important role in ovarian differentiation in the early development of the female gonad in mammals and fish. How EDCs affect Foxl2 expression is little known. In this study, we isolated a Foxl2 cDNA from the ovary of rare minnow Gobiocypris rarus and examined its expression during early development stages and in different adult tissues. Then, we analyzed Foxl2 expression in G. rarus juvenile following 3-day exposure to 17α- ethinylestradiol (EE2), 4-n-nonylphenol (NP), and bisphenol A (BPA). Alignment of known Foxl2 sequences among vertebrates showed high identity in forkhead domain and C-terminal region with other vertebrate proteins. Quantitative RT-PCR analysis showed that Foxl2 expression was linear decrease and cyp19a1a, the downstream target gene of Foxl2, had no correlation with Foxl2 from 18 to 50 days post fertilization (dpf). Among different adult tissues, Foxl2 is mainly expressed in ovary, brain, gill, eye, and male spleen. In the 3-day exposure, the juvenile fish to EDCs, 0.1 nM EE2, and 1 nM BPA significantly up-regulated the expression of Foxl2 gene, while NP had no effect on Foxl2 expression. Altogether, these results provide basic data for further study on how Foxl2 mediates EDCs impact on the sexual differentiation in G. rarus.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis.

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDSIKK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F. chinensis.