In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 µM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400 µM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

In Vitro and In Vivo Metabolic Activation and Hepatotoxicity of Environmental Pollutant 2,6-Dimethylphenol.

2,6-Dimethylphenol (2,6-DMP) is an environmental pollutant found in industrial wastewater. Exposure to 2,6-DMP is of increasing concern as it endangered reportedly some aquatic animals. In this study, we investigated the metabolic activation and hepatotoxicity of 2,6-DMP. 2,6-DMP was metabolized to an o-quinone methide intermediate in vitro and in vivo. The electrophilic metabolite was reactive to the sulfhydryl groups of glutathione, N-acetyl cysteine, and cysteine. NADPH was required for the formation of the reactive metabolite. The quinone methide intermediate reacted with cysteine residues to form hepatic protein adduction. A single dose of 2,6-DMP induced marked elevation of serum ALT and AST in mice. Both the protein adduction and hepatotoxicity of 2,6-DMP showed dose dependency.

Metabolic Activation of Gemfibrozil Mediated by Cytochrome P450 Enzymes and Sulfotransferases.

Gemfibrozil (GEM), a lipid regulator, is a fibric acid derivative widely used in the treatment of hyperlipidemia. It has been reported that GEM can induce acute liver injury in the course of therapy in clinical practice, so it is necessary to elucidate the mechanisms of toxic action. The present study focused on metabolic activation of GEM, possibly participating in GEM-mediated liver injury. A benzylic alcohol metabolite (M1), along with a phenol metabolite (M2), was detected in microsomal incubations, rat primary hepatocyte culturing, and rats given GEM. A GSH conjugate (M3) was detected in cultured rat hepatocytes after exposure to GEM. Formation of M1 was found to be NADPH dependent, and generation of M3 required M1 and 3'-phosphoadenosine-5'-phosphosulfate. It is most likely that GEM was biotransformed to M1, which was further metabolized to a sulfate. The resulting sulfate was reactive to bio-thiols. Cytochrome P450 and sulfotransferases participated in the phase I and phase II reactions, respectively. M1 and M3 were chemically synthesized, and their structures were characterized by mass spectrometry and NMR. The present study has particular value for elucidating the mechanism of liver injury caused by GEM.

Effects of different photoperiods on flower opening, flower closing and circadian expression of clock-related genes in Iris domestica and I. dichotoma.

The circadian clock can entrain to forced light-dark cycles by adjusting the phases and periods of flower opening and closing in ephemeral flowers. The responses of circadian rhythms to the same light conditions differ from species. However, the differences in internal genetic mechanisms underlying the different responses between species remain unclear. Iris domestica and I. dichotoma have ephemeral flowers and significantly divergent flower opening and closing times. The effects of different photoperiods (continuous darkness, 4L20D, 8L16D, 12L12D, 16L8D, 20L4D and continuous white light) on flower opening and closing, and expression patterns of seven genes (CRYPTOCHROME 1, PHYTOCHROME B, LATE ELONGATED HYPOCOTYL, PSEUDO RESPONSE REGULATOR 95, PHYTOCHROME INTERACTING FACTOR 4-like, SMUX AUXIN UP RNA 64-like and senescence-associated gene 39-like) involved in the circadian regulation of flower opening and closing were compared between I. domestica and I. dichotoma. Flower opening and closing in the two species exhibited circadian rhythms under continuous darkness (DD), but showed arrhythmia in continuous white light (LL). In the two species, keeping robust rhythms, strong synchronicity, rapid progressions of flower opening and closing and reaching full opening stage required a dark period longer than 4 h. In light-dark cycles with dark periods longer than 4 h, flower opening and closing times of the two species delayed with the delay of dawn, and the degree to which flower opening time varies with the time of dawn was greater in I. dichotoma than in I. domestica. The arrhythmia of flower opening and closing under 20L4D and LL would result from the arrhythmic output signals rather than arrhythmia of oscillators and photoreceptors. The different responses of the two species to the change of photoperiods would be caused by the transcriptional differences of genes in the output pathway of circadian clock system rather than in the input pathway or oscillators.

To bloom once or more times: the reblooming mechanisms of Iris germanica revealed by transcriptome profiling.

BACKGROUND: The reblooming bearded iris (Iris germanica) can bloom twice a year, in spring and autumn. The extended ornamental period makes it more popular and brings additional commercial values. However, little is known about the reblooming mechanisms, making the breeding programs time-consuming and labor-wasting. Therefore, a comparative transcriptome profiling was conducted on once-bloomers and rebloomers from the same F1 generation on six development stages, and the candidate genes associated with reblooming were identified. RESULTS: A total of 100,391 unigenes were generated, the mean length being 785 bp. In the three comparisons (the floral initiation stage of spring flowering in once-bloomers (OB-T1) vs the floral initiation stage of spring flowering in rebloomers (RB-T1); RB-T1 vs the floral initiation stage of autumn flowering in rebloomers (RB-T5); OB-T1 vs RB-T5), a total of 690, 3515 and 2941 differentially expressed genes (DEGs) were annotated against the public databases, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis focused on the photoperiod response, the temperature insensitivity and the growth speed, to remove the redundant DEGs and figure out the candidate key genes. As a result, the following four genes, PHYTOCHROME A (PHYA), GIGANTEA (GI), SHORT VEGETATIVE PERIOD (SVP) and AUXIN RESPONSE FACTOR (ARF), were considered to be involved in the second floral initiation of the rebloomers. CONCLUSION: This research provides valuable information for the discovery of the reblooming-related genes. The insights into the molecular mechanisms of reblooming may accelerate the breeding of bearded iris and other perennials.

Functional characterization of two flowering repressors SHORT VEGETATIVE PHASE and TERMINAL FLOWER 1 in reblooming bearded Iris (Iris spp.).

Reblooming bearded iris (Iris spp.) could bloom in both spring and autumn, which has extended the ornamental periods. Our previous transcriptome analysis has indicated the possible regulatory role of SHORT VEGETATIVE PHASE (SVP) in reblooming of bearded iris. Moreover, it has been revealed that the mutations of TERMINAL FLOWER 1 (TFL1) led to the continuous-flowering phenotypes in rose (Rosa spp.) and strawberry (Fragaria spp.). In order to verify the functions of these two genes on reblooming in bearded iris, IgSVP and IgTFL1 were isolated and functionally characterized. All the overexpression Arabidopsis lines of IgSVP and IgTFL1 generated the late-flowering phenotypes, indicating their functions as flowering repressors. The ectopic expression of IgSVP and IgTFL1 also generated phenotypic changes on flowers, inflorescences and branch structures. Moreover, the protein-protein interaction was found between a homologue of IgSVP and the floral meristem identity gene APETALA 1. The expression profiling showed that IgSVP was expressed significantly lower in the rebloomers in the second floral initiation stage (T5) than those of the first one (T1) in both the once-bloomers and the rebloomers, suggesting the possible regulation of IgSVP on reblooming. However, the expression level of IgTFL1 in the rebloomers was significantly higher in T5 than that in T1. The functional characterization of the two important flowering repressors IgSVP and IgTFL1 could lay solid foundation for future molecular breeding of iris, for example, knocking out the key repressors by CRISPR/Cas9 system to extend the ornamental periods of bearded iris.

Metabolic Activation of Pesticide Isoprocarb Mediated by CYP3A4 and the Possible Correlation with Its Cytotoxicity.

Isoprocarb (IPC), one of the most important carbamate pesticides, is used to control pests, such as rice planthoppers in crops. Studies have found that IPC induced hepatotoxicity in poultry chicken. However, the mechanisms of IPC-induced hepatotoxicity are unclear. The objectives of this study were to characterize reactive metabolites of IPC in vitro and in vivo, to identify cytochrome P450 enzymes for metabolic activation, and to define a possible correlation between the metabolic activation and cytotoxicity of IPC. In GSH- or NAC-supplemented microsomal incubations, one GSH conjugate (M6) and two NAC conjugates (M7 and M8) were detected after exposure to IPC. The corresponding GSH conjugate and NAC conjugates were found in the liver homogenates and urine of mice after IPC administration. IPC was found to be metabolized to a quinone intermediate reactive to GSH in vitro and in vivo. IPC was found to induce marked cytotoxicity in cultured mouse primary hepatocytes. Ketoconazole, a selective CYP3A4/5 enzyme inhibitor, attenuated the susceptibility of hepatocytes to IPC cytotoxicity.

Overexpressing the NAC transcription factor LpNAC13 from Lilium pumilum in tobacco negatively regulates the drought response and positively regulates the salt response.

NACs are one of the largest transcription factor families in plants and are involved in the response to abiotic stress. A new stress-responsive NAC transcription factor gene, LpNAC13, was isolated from Lilium pumilum bulbs. The expression of LpNAC13 was induced by drought, salt, cold and ABA treatments. LpNAC13 overexpressing plants were generated to explore the function of LpNAC13 in response to drought and salt stress. Overexpression of LpNAC13 in tobacco displayed a reduced drought tolerance but exhibited an enhanced salt tolerance. The LpNAC13 overexpression plants had decreased antioxidant enzyme activities, content of proline and chlorophyll, increased MDA content under drought condition, the results in the LpNAC13 plants under salt condition were opposite to those under drought condition. The seed germination and root length assays of overexpression of LpNAC13 showed decreased sensitivity to ABA. Functional analyses demonstrate that LpNAC13 plays opposite roles in drought and salt stress tolerance, acting as a negative regulator of drought response but as a positive regulator of salt response in tobacco.