Efficient identification of trait-associated loss-of-function variants in the UK Biobank cohort by exome-sequencing based genotype imputation.

The large-scale open access whole-exome sequencing (WES) data of the UK Biobank ~200,000 participants is accelerating a new wave of genetic association studies aiming to identify rare and functional loss-of-function (LoF) variants associated with complex traits and diseases. We proposed to merge the WES genotypes and the genome-wide genotyping (GWAS) genotypes of 167,000 UKB homogeneous European participants into a combined reference panel, and then to impute 241,911 UKB homogeneous European participants who had the GWAS genotypes only. We then used the imputed data to replicate association identified in the discovery WES sample. The average imputation accuracy measure r2 is modest to high for LoF variants at all minor allele frequency intervals: 0.942 at MAF interval (0.01, 0.5), 0.807 at (1.0 × 10-3 , 0.01), 0.805 at (1.0 × 10-4 , 1.0 × 10-3 ), 0.664 at (1.0 × 10-5 , 1.0 × 10-4 ) and 0.410 at (0, 1.0 × 10-5 ). As applications, we studied associations of LoF variants with estimated heel BMD and four lipid traits. In addition to replicating dozens of previously reported genes, we also identified three novel associations, two genes PLIN1 and ANGPTL3 for high-density-lipoprotein cholesterol and one gene PDE3B for triglycerides. Our results highlighted the strength of WES based genotype imputation as well as provided useful imputed data within the UKB cohort.

Targeting Neuronal GPR65 With Delayed BTB09089 Treatment Improves Neurorehabilitation Following Ischemic Stroke.

BACKGROUND: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown. METHODS: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65-/-) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65-/- and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting. RESULTS: BTB09089 significantly promoted motor outcomes in WT but not in GPR65-/- mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65-/- mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65-/- mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65-/- mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice. CONCLUSIONS: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.

Predictors of anxiety among pregnant women with gestational diabetes mellitus and their partners: The mediating role of marital satisfaction.

AIMS: This study aims to examine the prevalence of anxiety symptoms and identify predictors of anxiety among pregnant women with gestational diabetes mellitus and their partners and explore the mediating role of marital satisfaction between maternal and paternal anxiety. DESIGN: A cross-sectional study was conducted in Guangzhou, China, from July 2021 to May 2022. METHODS: A total of 306 dyads of pregnant women with gestational diabetes mellitus and their partners completed the State-Trait Anxiety Inventory, Locke-Wallace Marital Adjustment Test and the socio-demographic and clinical data sheet. RESULTS: The prevalence of anxiety symptoms was 32.4% and 36.6% in pregnant women with gestational diabetes mellitus and their partners, respectively. The predictors of maternal anxiety were paternal anxiety, maternal marital satisfaction, maternal monthly salary, fasting glucose value and 1-h glucose value. By contrast, the predictors of paternal anxiety were maternal anxiety, paternal marital satisfaction and paternal monthly salary. Moreover, the relationship between maternal and paternal anxiety was mediated by marital satisfaction. CONCLUSIONS: The anxiety symptoms of pregnant women with gestational diabetes mellitus and their partners influence each other, and this relationship was mediated by marital satisfaction. Every couple should be screened for anxiety symptoms and treated as a team rather than focusing solely on the pregnant woman.

[Pharmacodynamic substances and therapeutic potential of Wuji Pills:based on UPLC-Q-TOF-MS/MS and network pharmacology].

As a classic prescription, Wuji Pills is composed of Coptidis Rhizoma, Euodiae Fructus Preparata, and stir-fried Paeo-niae Radix Alba at the ratio of 6∶1∶6. The practical application of it is limited compared with other famous Chinese medicine prescriptions. Only one company produces Wuji Pills in China. In this study, ultra-performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to analyze and identify 26 identical compounds from Wuji Pills and drug-containing plasma of rats. Based on these components, 46 potential targets were screened out with network pharmacology methods, followed by the component-target network construction, Gene Ontology(GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and disease prediction. It was concluded that Wuji Pills acted on core targets such as PTGS2, PTSG1, NCOA2, HSP9 OAD1, and RXRA through magnoflorine, hydroxyevodiamine, daucosterol, and berberine and exerted pharmacodynamic effects through various pathways such as calcium ion signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3 K-Akt) signaling pathway, and vascular endothelial growth factor(VEGF) signaling pathway. Thus, Wuji Pills has therapeutic potential for Alzheimer's disease, diabetes mellitus, myocardial ischemia, and other diseases in addition to the conventional disease(irritable bowel syndrome, IBS). The above research results can provide a reference for the comprehensive interpretation of the pharmacodynamic basis of Wuji Pills and the expansion of clinical application. At the same time, a lot of components in serum and the in vivo transformed and metabolized components of Wuji Pills have similar structure and relative molecular weight. In theory, these components may show additive effects and the competitive/antagonistic effects on the same target. According to the hypothesis of "additive effect of multiple components for a single target" in traditional Chinese medicine, multiple similar components may exert the additive effects on local targets. This study can partly prove the scientificity of this hypothesis and provide laboratory evidence.

Curcuma's extraction attenuates propranolol-induced psoriasis like in mice by inhibition of keratin, proliferating cell nuclear antigen and toll-like receptor expression.

Curcuma was the dried rhizomes of Curcuma kwangsiensis S.G. Lee et C.F. Liang (Chinese name: e zhu), have been used in China for thousands of years. There are some reports have shown that curcumin, the major component of curcuma, has a good curative effect on psoriasis, but the mechanism is still unknown, so the present study was designed to investigate the effect of curcuma's extraction on psoriasis-like mouse, and to explore the mechanisms of therapy. First, we observed that curcuma's extractions effect on mitosis of mouse vaginal epithelial cells; then making psoriasis like model and measuring the score of skin damage on days 7 and 14; finally, we observed the expression of immune factors (CK14, CK16, CK17, PCNA, TLR-2, TLR-4, TLR-9) in propranolol induced psoriasis like rats. Curcuma's extraction prohibited the mitosis of mouse vaginal epithelial cells; curcuma's extractions have a significantly efficacy and dose dependent inhibition on imiquimod induced psoriasis like rats; and the expression of immune factors (CK14, CK16, CK17, PCNA, TLR-2, TLR-4, TLR-9) was decreasing in the curcuma's extraction treated groups compared with normal groups. Our research proved that curcuma's extractions have a significantly efficacy on psoriasis like rats, thus, curcuma's extractions can be a potential novel treatment for psoriasis. Furthermore, the expression of immune factors was decreasing after treatment with curcuma's extraction suggest us cytokines has strong relation with the mechanism of therapy for psoriasis. Our results contribute towards validation of curcuma in the treatment of psoriasis and other joint disorders.

In vitro and in vivo metabolic profiles of fasiglifam using ultrahigh-performance liquid chromatography combined with Q-Exactive Orbitrap tandem mass spectrometry.

RATIONALE: Fasiglifam is an orally available and selective partial agonist of hGPR40 receptor, which was unexpectedly terminated at phase III clinical trials due to its severe hepatotoxicity. To fully understand the mechanism of action of fasiglifam, it is necessary to investigate its in vitro and in vivo metabolic profiles. METHODS: For in vitro metabolism, fasiglifam was incubated with rat or human liver microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate tetrasodium salt, glutathione (GSH) and uridine diphosphate glucuronic acid trisodium salt for 60 min. For in vivo metabolism, fasiglifam was orally administered to rats at a single dose of 20 mg/kg and the bile was collected. In vitro and in vivo samples were analyzed by the developed ultrahigh-performance liquid chromatography combined with Q-Exactive Orbitrap tandem mass spectrometry. The structures of metabolites were proposed according to their accurate masses and fragment ions. RESULTS: A total of eight metabolites, including an acyl-GSH adduct, were detected and identified. M1 (acylglucuronide) and M5 (carboxylic acid derivative) were the major metabolites of fasiglifam. Metabolic pathways of fasiglifam involved oxygenation, oxidative dealkylation, dehydrogenation, glucuronidation and GSH conjugation. Fasiglifam may undergo metabolic bioactivation via acylglucuronide. CONCLUSIONS: Oxidative dealkylation and glucuronidation were the predominant metabolic pathways of fasiglifam in vitro and in vivo. Metabolic bioactivation via acylglucuronide may be the perpetrator of its hepatotoxicity. Our findings would be helpful in understanding the disposition of fasiglifam as well as its hepatotoxicity.

A validated LC-MS/MS method for the determination of senkyunolide I in dog plasma and its application to a pharmacokinetic and bioavailability studies.

Senkyunolide I is one of the major bioactive components in the herbal medicine Ligusticum chuanxiong. The aim of this study was to develop and validate a fast, simple and sensitive LC-MS/MS method for the determination of senkyunolide I in dog plasma. The plasma samples were processed with acetonitrile and separated on a Waters Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm). The mobile phase consisted of 0.1% formic acid aqueous and acetonitrile was delivered at a flow rate of 0.3 mL min-1 . The detection was achieved in the positive selected reaction monitoring mode with precursor-to-product transitions at m/z 225.1 → 161.1 for senkyunolide I and at m/z 349.1 → 305.1 for an internal standard. The assay was linear over the tested concentration range, from 0.5 ng mL-1 to 1000 ng mL-1 , with a correlation coefficient >0.9992. The mean extraction recovery from dog plasma was within the range of 85.78-93.25%, while the matrix effect of the analyte was within the range of 98.23-108.89%. The intra- and inter-day precisions (RSD) were <12.12% and the accuracy (RR) ranged from 98.89% to 104.24%. The validated assay was successfully applied to pharmacokinetic and bioavailability studies of senkyunolide I in dogs. The results demonstrated that (a) senkyunolide I showed short elimination half-life (<1 h) in dog, (b) its oral bioavailability was >40% and (c) senkyunolide I showed dose-independent pharmacokinetic profiles in dog plasma over the dose range of 1-50 mg kg-1 .

Simultaneous determination of 20(S)-protopanaxadiol and its three metabolites in rat plasma by LC-MS/MS: application to their pharmacokinetic studies.

The aim of this study was to develop an LC-MS/MS method for simultaneous determination of 20(S) protopanaxadiol (PPD) and its three metabolites, PPD-glucuronide (M1), (20S,24S)-epoxy-dammarane-3,12,25-triol (M2) and (20S,24R)-epoxydammarane-3,12,25-triol (M3), in rat plasma. Precipitation with acetonitrile was employed for sample preparation and chromatographic separations were achieved on a C18 column. The sample was detected using triple quadrupole tandem mass spectrometer with selected reaction monitoring mode. The monitored precursor-to-product ion transitions were m/z 459.4 → 375.3 for PPD, m/z 635.4 → 113.0 for M1, m/z 477.4 → 441.4 for M2 and M3 and m/z 475.4 → 391.3 for IS. The developed assay was validated according to the guidelines of the US Food and Drug Administration. The calibration curves showed good linearity over the tested concentration ranges (r > 0.9993), with the LLOQ being 1 ng/mL for all analytes. The intra- and inter-day precisions (RSD) were < 9.51% while the accuracy (RE) ranged from -8.91 to 12.84%. The extraction recovery was >80% and no obvious matrix effect was detected. The analytes were stable in rat plasma with the RE ranging from -12.34 to 9.77%. The validated assay has been successfully applied to the pharmacokinetic study of PPD as well as its metabolites in rat plasma. According to the pharmacokinetic parameters, the in vivo exposures of M1, M2 and M3 were 11.91, 47.95 and 22.62% of that of PPD, respectively.

The terrestrial end-Permian mass extinction in the paleotropics postdates the marine extinction.

The end-Permian mass extinction was the most severe ecological event during the Phanerozoic and has long been presumed contemporaneous across terrestrial and marine realms with global environmental deterioration triggered by the Siberian Traps Large Igneous Province. We present high-precision zircon U-Pb geochronology by the chemical abrasion-isotope dilution-thermal ionization mass spectrometry technique on tuffs from terrestrial to transitional coastal settings in Southwest China, which reveals a protracted collapse of the Cathaysian rainforest beginning after the onset of the end-Permian marine extinction. Integrated with high-resolution geochronology from coeval successions, our results suggest that the terrestrial extinction occurred diachronously with latitude, beginning at high latitudes during the late Changhsingian and progressing to the tropics by the early Induan, spanning a duration of nearly 1 million years. This latitudinal age gradient may have been related to variations in surface warming with more degraded environmental conditions at higher latitudes contributing to higher extinction rates.

Lanostane triterpenoids from the fungus Physisporinus vitreus and their inhibitory activity against nitric oxide production.

Eight undescribed lanostane triterpenoids, physivitrins A-H, along with four known analogues, were isolated from cultures of the fungus Physisporinus vitreus. Their structures were elucidated on the basis of extensive spectroscopic methods, in which the absolute configuration of physivitrin A was elucidated using electronic circular dichroism calculation and nuclear magnetic resonance (NMR) calculation with DP4+ analysis. Physivitrins B and C showed inhibitory activities against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages with IC50 values of 7.5 and 23.5 µM, respectively. Meanwhile, proinflammatory cytokines (TNF-α, iNOS and IL-1ß) mRNA expression was also inhibited by physivitrin B significantly.

Prevalence, distribution, accumulation, and risk of environmental corticosteroids and estrogens in biofilms from the Pearl River Delta.

Biofilms play a significant role in the biogeochemical processing of organic matter and the environmental fate of emerging pollutants. In this study, we investigated the occurrence and distribution of 32 endocrine-disrupting chemicals (EDCs), including 24 environmental corticosteroids (ECs) and 8 environmental estrogens (EEs), in natural biofilms from the Pearl River system. Their association between biofilms and water and environmental risk were assessed. The ECs and EEs ubiquitously occurred in the biofilms, ranging from <0.61-6.57 ng/g and <0.8-2535 ng/g, respectively. Temporally, there was no obvious variance in either ECs or EEs in the biofilms during the winter and summer, and their concentrations exhibited a spatial trend of upward to midstream, descending downstream, and then seaward attenuation at the estuary. For ECs and EEs, the similar levels of field-derived bioconcentration factors (BCFs) (logarithm values: 2.42-2.86 and 2.72-2.98, respectively) and biofilm organic carbon-normalized partitioning coefficients (Kboc) (3.39-3.69 and 3.35-3.95) suggest the comparable potential of accumulation and sorption by biofilms between these two classes of EDCs. In addition, higher values of BCF and Kboc for the EEs were found in winter and were correspondingly comparable to their distribution coefficients (Kd) and Koc derived from suspended particles and sediment, revealing that biofilms are a competitive environmental compartment for capturing EDCs, particularly during the mature period. A positive logKboc-logKow relationship suggests hydrophobic partitioning as a primary interaction mechanism between the biofilm and EEs. Moreover, high risks from biofilm-associated ECs and EEs might have posed to the fluvial ecosystem. This study provides original insights into the occurrence, fate, and risk of ECs in natural biofilms for the first time and demonstrates that biofilms may not only serve as reservoirs but also serve as sentinels for fluvial EDC contamination. These results contribute to the further understanding of the behavior and fate of EDCs in aquatic environments.

KMT2E Haploinsufficiency Manifests Autism-Like Behaviors and Amygdala Neuronal Development Dysfunction in Mice.

Autism spectrum disorders (ASD) are highly heterogeneous neurodevelopmental disorders characterized by impaired social interaction skills. Whole exome sequencing has identified loss-of-function mutations in lysine methyltransferase 2E (KMT2E, also named MLL5) in ASD patients and it is listed as an ASD high-risk gene in humans. However, experimental evidence of KMT2E in association with ASD-like manifestations or neuronal function is still missing. Relying on KMT2E+/- mice, through animal behavior analyses, positron emission tomography (PET) imaging, and neuronal morphological analyses, we explored the role of KMT2E haploinsufficiency in ASD-like symptoms. Behavioral results revealed that KMT2E haploinsufficiency was sufficient to produce social deficit, accompanied by anxiety in mice. Whole-brain 18F-FDG-PET analysis identified that relative amygdala glycometabolism was selectively decreased in KMT2E+/- mice compared to wild-type mice. The numbers and soma sizes of amygdala neurons in KMT2E+/- mice were prominently increased. Additionally, KMT2E mRNA levels in human amygdala were significantly decreased after birth during brain development. Our findings support a causative role of KMT2E in ASD development and suggest that amygdala neuronal development abnormality is likely a major underlying mechanism.

Suspended particulate matter-associated environmental corticosteroids in the Pearl River, China: Occurrence, distribution, and partitioning.

Suspended particulate matter (SPM) plays an important role in the geochemical behavior and fate of organic micropollutants in aquatic environments. However, the presence of trace emerging endocrine disruptors such as environmental corticosteroids (ECs) in SPM is less well understood. This study focused on the occurrence, distribution, and partitioning of SPM-associated ECs in the Pearl River system, China. Ubiquitous particulate ECs were found in the surface water of the rivers at average concentrations (dry weight) between 0.46 ng/g (flumethasone) and 8.83 ng/g (clobetasone butyrate). The total EC (∑ECs) concentrations of the 24 selected target compounds varied from <1.03 ng/g to 62.3 ng/g, with an average and median of 17.6 ng/g and 13.7 ng/g, respectively. Higher SPM-bound EC levels were commonly observed in winter (dry season), and spatially, their relatively high contamination in urban tributary networks decreased while flowing to mainstreams and then gradually attenuated from upstream to the estuary. Despite the approximately 90 % mass distribution of ∑ECs in the aqueous phase, approximately 50 % of their effect burden was derived from the suspended particulate fractions. For the first time, in situ SPM-water partitioning coefficients (Kp) and their organic carbon-normalized ones (Koc) of ECs were determined in surface waters, and a field-derived preliminary linear equation was proposed to estimate Koc for ECs using basic physicochemical parameters n-octanol/water partitioning coefficient (Kow), which is of importance with regard to the assessment of transport, fate, and risk of these emerging hazardous chemicals. Furthermore, the significant logKoc-logKow relationship for ECs reveals that nonspecific hydrophobic partitioning is a major association mechanism between SPM and ECs. Moreover, hydrogen bonding is suggested to be a prevailing specific binding mechanism and provides more contribution to nonhydrophobic interactions between ECs and particulate organic matter than environmental estrogens.

Delayed CO2 postconditioning promotes neurological recovery after cryogenic traumatic brain injury by downregulating IRF7 expression.

AIMS: Few treatments are available in the subacute phase of traumatic brain injury (TBI) except rehabilitation training. We previously reported that transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects against cerebral ischemia/reperfusion injury. In this study, it was hypothesized that delayed CO2 postconditioning (DCPC) starting at the subacute phase may promote neurological recovery of TBI. METHODS: Using a cryogenic TBI (cTBI) model, mice received DCPC daily by inhaling 5%/10%/20% CO2 for various time-courses (one/two/three cycles of 10-min inhalation/10-min break) at Days 3-7, 3-14 or 7-18 after cTBI. Beam walking and gait tests were used to assess the effect of DCPC. Lesion size, expression of GAP-43 and synaptophysin, amoeboid microglia number and glia scar area were detected. Transcriptome and recombinant interferon regulatory factor 7 (Irf7) adeno-associated virus were applied to investigate the molecular mechanisms. RESULTS: DCPC significantly promoted recovery of motor function in a concentration and time-course dependent manner with a wide therapeutic time window of at least 7 days after cTBI. The beneficial effects of DCPC were blocked by intracerebroventricular injection of NaHCO3 . DCPC also increased puncta density of GAP-43 and synaptophysin, and reduced amoeboid microglia number and glial scar formation in the cortex surrounding the lesion. Transcriptome analysis showed many inflammation-related genes and pathways were altered by DCPC, and Irf7 was a hub gene, while overexpression of IRF7 blocked the motor function improvement of DCPC. CONCLUSIONS: We first showed that DCPC promoted functional recovery and brain tissue repair, which opens a new therapeutic time window of postconditioning for TBI. Inhibition of IRF7 is a key molecular mechanism for the beneficial effects of DCPC, and IRF7 may be a potential therapeutic target for rehabilitation after TBI.

Comparative Analysis of Aristolochic Acids in Aristolochia Medicinal Herbs and Evaluation of Their Toxicities.

Aristolochic acids (AAs) are a group of nitrophenanthrene carboxylic acids present in many medicinal herbs of the Aristolochia genus that may cause irreversible hepatotoxicity, nephrotoxicity, genotoxicity and carcinogenicity. However, the specific profile of AAs and their toxicity in Aristolochia plants, except for AAs Ι and ΙΙ, still remain unclear. In this study, a total of 52 batches of three medicinal herbs belonging to the Aristolochia family were analyzed for their AA composition profiles and AA contents using the UPLC-QTOF-MS/MS approach. The studied herbs were A. mollissima Hance (AMH), A. debilis Sieb.etZucc (ADS), and A. cinnabaria C.Y.Cheng (ACY). Chemometrics methods, including PCA and OPLS-DA, were used for the evaluation of the Aristolochia medicinal herbs. Additionally, cytotoxicity and genotoxicity of the selected AAs and the extracts of AMH and ADS were evaluated in a HepG2 cell line using the MTT method and a Comet assay, respectively. A total of 44 AAs, including 23 aristolochic acids and 21 aristolactams (ALs), were detected in A. mollissima. Moreover, 41 AAs (23 AAs and 18 ALs) were identified from A. debilis Sieb, and 45 AAs (29 AAs and 16 ALs) were identified in A. cinnabaria. Chemometrics results showed that 16, 19, and 22 AAs identified in AMH, ADS, and ACY, respectively, had statistical significance for distinguishing the three medicinal herbs of different origins. In the cytotoxicity assay, compounds AL-BΙΙ, AAΙ and the extract of AMH exhibited significant cytotoxicities against the HepG2 cell line with the IC50 values of 0.2, 9.7 and 50.2 µM, respectively. The results of the Comet assay showed that AAΙ caused relatively higher damage to cellular DNA (TDNA 40-95%) at 50 µM, while AAΙΙ, AMH and ADS extracts (ranged from 10 to 131 µM) caused relatively lower damage to cellular DNA (TDNA 5-20%).

Evaluation of the Direct and Indirect Regulation Pathways of Glutathione Target to the Hepatotoxicity of Microcystin-LR.

Glutathione (GSH) plays crucial roles in regulating the hepatotoxicity of Microcystin-LR (MCLR) by inhibiting oxidative stress or by toxin conjugation. Based on MCLR conjugation product preparation and purification, the direct and indirect regulation pathways for GSH were fully evaluated. Protein phosphatase inhibition analysis verified that GSH conjugation was an effective pathway to regulate the inhibition effect of MCLR, while GSH had slight influence on the toxicity of MCLR. Research on oxidative stress showed that both regulation pathways could reduce the formation of reactive oxygen species (stimulated by MCLR and regulated by NADH oxidase) and regulate the adverse effects on antioxidant enzymes. By evaluating the contributions for both pathways, it could be found that the indirect pathway had significant contribution to eliminating cellular reactive oxygen species and regulating protein phosphatases inhibition, while the direct regulation pathway had moderate influence. As glutathione transferases facilitated the transformation of MCLR, the hepatotoxicity of MCLR could be effectively regulated by GSH conjugation pathway, especially with abundant exogenous GSH.