Morphological and phylogenetic analyses reveal a new species of Anthracophyllum (Omphalotaceae, Agaricales) in Zhejiang Province, China.

During the investigations of macrofungi resources in Zhejiang Province, China, an interesting wood rot fungus was collected. Based on morphological and molecular phylogenetic studies, it is described as a new species, Anthracophyllum sinense. A. sinense is characterized by its sessile, charcoal black and pleurotoid pileus, sparse lamellae occasionally branching, clavate basidia with long sterigmata [(3-)6-7(-8) µm], and non-heteromorphous cystidia. A. sinense establishes a separate lineage close to A. archeri and A. lateritium in the phylogenetic tree.

Circulating circRNA predicting the occurrence of hepatocellular carcinoma in patients with HBV infection.

A microarray-based high-throughput screening of human circulating circular RNA (circRNA) was applied with five patients newly diagnosed with hepatocellular carcinoma (HCC), five patients with HBV-positive chronic hepatitis (CH) and five healthy controls (NC) enrolled. The plasma of HCC patients after hepatectomy was also collected. After multiple staged validation, we obtained five circRNAs as candidate. Based on the stratified risk score analysis, three increased circRNAs including circ_0009582, circ_0037120 and circ_0140117 were confirmed as candidate circulating fingerprints for distinguishing HCC from CH or NC group. With the combination of AFP, higher sensitivity and specificity were further guaranteed, suggesting that circ_0009582, circ_0037120 and circ_0140117 may serve as potential biomarkers for predicting the occurrence of HCC in patients with HBV infection.

CD97 Promotes Tumor Aggressiveness Through the Traditional G Protein-Coupled Receptor-Mediated Signaling in Hepatocellular Carcinoma.

Cluster of differentiation 97 (CD97) is a member of the epidermal growth factor seven-transmembrane family belonging to the class B G protein-coupled receptors (GPCRs). The protein affects tumor aggressiveness through its cellular ligand CD55 stimulation and exhibits adhesive properties. Studies have demonstrated the involvement of CD97 in dedifferentiation, migration, invasiveness, and metastasis of tumors. However, little information is currently available on the specific role of CD97 in hepatocellular carcinoma (HCC). Here, we have shown that CD97 up-regulation in HCCs is positively correlated with tumor metastasis. Functionally, CD97 promoted cell migration and invasion in vitro. In an in vivo mouse model, overexpression of CD97 in HCC cells led to accelerated lung metastasis. Mechanistically, CD97 cooperated with the altered regulator, GPCR kinase 6 (GRK6), to mediate GPCR desensitization and internalization. Down-regulation of GRK6 suppressed CD97 internalization and promoted CD97 expression. Integrated regulatory interactions between CD97 and GRK6 stimulated downstream matrix metalloproteinase 2/9 secretion and, consequently, HCC metastasis. Conclusion: Our collective findings support the utility of CD97 as an effective potential prognosticator and therapeutic target for HCC.

Single and double boron atoms doped nanoporous C2N-h2D electrocatalysts for highly efficient N2 reduction reaction: a density functional theory study.

The electrocatalytical process is the most efficient way to produce ammonia (NH3) under ambient conditions, but developing a highly efficient and low-cost metal-free electrocatalysts remains a major scientific challenge. Hence, single atom and double boron (B) atoms doped 2D graphene-like carbon nitride (C2N-h2D) electrocatalysts have been designed (B@C2N and B2@C2N), and the efficiency of N2 reduction reaction (NRR) is examined by density functional theory calculation. The results show that the single and double B atoms can both be strongly embedded in natural nanoporous C2N with superior catalytic activity for N2 activation. The reaction mechanisms of NRR on the B@C2N and B2@C2N are both following an enzymatic pathway, and B2@C2N is a more efficient electrocatalyst with extremely low overpotential of 0.19 eV comparing to B@C2N (0.29 eV). In the low energy region, the hydrogenation of N2 is thermodynamically more favorable than the hydrogen production, thereby improving the selectivity for NRR. Based on these results, a new double-atom strategy may help guiding the experimental synthesis of highly efficient NRR electrocatalysts.

The aberrant expression of MEG3 regulated by UHRF1 predicts the prognosis of hepatocellular carcinoma.

MEG3 as a tumor suppressor has been reported to be linked with pathogenesis of malignancies including hepatocellular carcinoma (HCC). However, the mechanism of MEG3 in HCC still remains unclear. In our study, the aberrant decreased level of MEG3 in 72 tumor tissues obtained from HCC patients and cell lines was examined by using real-time PCR. The inhibition affection in proliferation and inducing affection in apoptosis was further confirmed in vivo and vitro, we also demonstrated that MEG3 regulates HCC cell proliferation and apoptosis partially via the accumulation of p53. Besides, the hypermethylation of MEG3 in promoter region was identified by bisulfite sequencing while MEG3 increased with the inhibition of methylation. Subsequently, UHRF1, a new identified oncogene which is required for DNA methylation and recruits, was investigated. A negative correlation of MEG3 and UHRF1 expression was verified in primary HCC tissues. Down-regulation of UHRF1 induced MEG3 expression in HCC cell lines, which could be reversed by the up-regulation of UHRF1. In addition, up-regulation of MEG3 in HCC cells partially diminished the promotion of proliferation induced by UHRF1. Moreover, Kaplan-Meier analysis demonstrated that the patients with low expression of MEG3 indicated worse overall and relapse-free survivals compared with high expression of MEG3. Cox proportional hazards analyses showed that MEG3 expression was an independent prognostic factor for HCC patients. In conclusion, we demonstrated MEG3, acting as a potential biomarker in predicting the prognosis of HCC, was regulated by UHRF1 via recruiting DNMT1 and regulated p53 expression.

S100A4 hypomethylation affects epithelial-mesenchymal transition partially induced by LMP2A in nasopharyngeal carcinoma.

To identify cellular target genes involved in NPC cell invasion and metastasis, gene expression profiles of CNE-1 cells with or without ectopic LMP2A expression were compared by using the metastatic gene array. S100 calcium binding protein A4 (S100A4) was the highest increased one among these genes both in mRNA and protein levels of NPC cells. Moreover, S100A4 was upregulated in LMP2A-positive NPC tissues. We found that CNE-1-S100A4 showed significantly increased invasion ability as compared to the controls both in vitro and in vivo, which indicated that S100A4 induced EMT occurrence and promoted metastasis. Notably, the DNA hypomethylation of S100A4 was found in LMP2A-positive NPC tissues. Besides, inhibition of DNA methyltransferases via 5-Aza-dC stimulated the expression of S100A4 in the cells without ectopic LMP2A expression. The methylation changes were confirmed by methylation specific PCR (MSP), suggesting that LMP2A ectopic expression led to the demethylation of S100A4 promoter. These results demonstrated that LMP2A-induced hypomethylation participated in regulating S100A4 expression in NPC. Our findings provide an evidence for the emerging notion that hypomethylation and activation of correlated genes are crucial for metastasis progression in cancer. © 2015 Wiley Periodicals, Inc.

PTPRO-associated hepatic stellate cell activation plays a critical role in liver fibrosis.

BACKGROUND/AIMS: PTPRO (protein tyrosine phosphatase, receptor type O) is implicated in diverse physiological and pathological processes in cancer and hepatic ischemia/reperfusion injury, although little is known about its role in hepatic fibrosis. METHODS: Here, by using genetically deficient mice, we reported that PTPRO knockout (PTPRO(-/-)) significantly attenuated liver injury, release of inflammatory factors, tissue remodeling, and liver fibrosis in two experimental mouse models of fibrogenesis induced by bile-duct ligation or carbon tetrachloride administration. RESULTS: However, we proved that PTPRO expression was strongly downregulated in clinical and experimental liver fibrosis specimens. Further investigations revealed that stimulation of primary hepatic stellate cells (HSCs) and hepatocytes with specific activator platelet-derived growth factor (PDGF)-BB increased PTPRO transcription in HSCs but had the opposite effect in primary hepatocytes. More importantly, synthetic short hairpin RNA targeting PTPRO significantly neutralized PDGF-BB-induced HSC proliferation and myofibroblast marker expression through downregulated phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. CONCLUSION: These observations confirm that PTPRO plays a critical role in liver fibrogenesis by affecting PDGF signaling in HSC activation and might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.

Norlignans from Pouzolzia zeylanica var. microphylla and their nitric oxide inhibitory activity.

Five new compounds, pouzolignan F [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (1), pouzolignan G [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (2), pouzolignan H [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butane] (3), pouzolignan I [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dime thoxyphenyl)-(4-hydroxy-3-methoxyphenyl)methyl]butane] (4), and pouzolignan J [1,4-dihydroxy-3-(3,5-dihydroxyphenyl) -2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butane] (5), along with two known compounds, indolyl-3-carboxylic acid (6) and uracil (7), were isolated from the aerial parts of Pouzolzia zeylanica (L.) Benn. var. microphylla (Wedd.) W.T.Wang. The structures of these compounds were characterized based on spectroscopic methods, including IR, NMR ((1)H-(1)H COSY, HSQC, HMBC, and NOESY), and HR-ESI/TOF-MS experiments. All the new norlignans were assayed for inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages.

Evaluating AI in medicine: a comparative analysis of expert and ChatGPT responses to colorectal cancer questions.

Colorectal cancer (CRC) is a global health challenge, and patient education plays a crucial role in its early detection and treatment. Despite progress in AI technology, as exemplified by transformer-like models such as ChatGPT, there remains a lack of in-depth understanding of their efficacy for medical purposes. We aimed to assess the proficiency of ChatGPT in the field of popular science, specifically in answering questions related to CRC diagnosis and treatment, using the book "Colorectal Cancer: Your Questions Answered" as a reference. In general, 131 valid questions from the book were manually input into ChatGPT. Responses were evaluated by clinical physicians in the relevant fields based on comprehensiveness and accuracy of information, and scores were standardized for comparison. Not surprisingly, ChatGPT showed high reproducibility in its responses, with high uniformity in comprehensiveness, accuracy, and final scores. However, the mean scores of ChatGPT's responses were significantly lower than the benchmarks, indicating it has not reached an expert level of competence in CRC. While it could provide accurate information, it lacked in comprehensiveness. Notably, ChatGPT performed well in domains of radiation therapy, interventional therapy, stoma care, venous care, and pain control, almost rivaling the benchmarks, but fell short in basic information, surgery, and internal medicine domains. While ChatGPT demonstrated promise in specific domains, its general efficiency in providing CRC information falls short of expert standards, indicating the need for further advancements and improvements in AI technology for patient education in healthcare.

RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma.

Background and Aims: Chronic hepatitis caused by hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC). We investigated the roles of oncogenic HBV infection-associated long noncoding RNAs in HCC. Methods: Bioinformatics analysis of data from the Cancer Genome Atlas (TCGA) was performed to screen potential oncogenic HBV-related lncRNAs. Next, we assessed their expression in clinical samples and investigated their correlation with clinical characteristics. The detailed oncogenic effects were analyzed by performing in vitro and in vivo studies. Results: RP11-40C6.2, an HBV infection-related lncRNA, was identified by analysis of the TCGA-Liver Hepatocellular Carcinoma database. Gene Set Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes revealed a strong association of RP11-40C6.2 with the Hippo signaling pathway. RP11-40C6.2 was overexpressed in HCC patients with HBV infection compared to those without HBV infection. RP11-40C6.2 transcription showed a positive association with HBV-X protein (HBx), but not HBV core protein (HBc) expression, both of which are carcinogenic proteins. Luciferase gene reporter and ChIP assays revealed that YAP1/TAZ/TEADs complex enhanced RP11-40C6.2 transcription by binding to its promoter area. RP11-40C6.2 showed oncogenic characteristics in HCC cell lines and in animal models that were mediated via activation of YAP1. In vitro ubiquitylation assay revealed that RP11-40C6.2 can promote the stabilization of YAP1 by stopping phosphorylation at its s127 residue and further stopping its degradation through binding to 14-3-3. Conclusions: RP11-40C6.2 is an HBV infection-related lncRNA that exerts its oncogenic effects by targeting the Hippo signaling pathway.

[Application of the theory of equal emphasis on muscle and bone in percutaneous vertebroplasty of lumbar osteoporotic compression fracture].

OBJECTIVE: To explore the clinical efficacy of percutaneous vertebroplasty(PVP) combined with nerve block in the treatment of lumbar osteoporotic vertebral compression fractures under the guidance of traditional chinese medicine "theory of equal emphasis on muscle and bone". METHODS: Total of 115 patients with lumbar osteoporotic vertebral compression fractures were treated by percutaneous vertebroplasty from January 2015 to March 2022, including 51 males and 64 females, aged 25 to 86 (60.5±15.9) years. Among them, 48 cases were treated with PVP operation combined with erector spinae block and joint block of the injured vertebral articular eminence (intervention group), and 67 cases were treated with conventional PVP operation (control group). The visual analogue scale(VAS) and Oswestry disability index(ODI) before operation, 3 days, 1 month and 6 months after operation between two groups were evaluated. The operation time, number of punctures and intraoperative bleeding between two groups were compared. RESULTS: The VAS and ODI scores of both groups improved significantly after operation compared with those before operation(P<0.05). Moreover, the VAS and ODI scores of 3 days and 1 month after operation of the intervention group improved more significantly than that of the control group(P<0.05). The difference of VAS and ODI scores before operation and 6 months after operation between two groups had no statistical significances(P>0.05). There was no statistically significant difference in the number of punctures and intraoperative bleeding between the two groups (P>0.05). CONCLUSION: Based on the theory of "equal emphasis on muscles and bones", PVP combined with nerve block can effectively relieve paravertebral soft tissue spasm and other "muscle injuries", which can significantly improve short-term postoperative low back pain and lumbar spine mobility compared to conventional PVP treatment, and accelerate postoperative recovery, resulting in satisfactory clinical outcomes.

Cancer-associated fibroblast exosomes promote chemoresistance to cisplatin in hepatocellular carcinoma through circZFR targeting signal transducers and activators of transcription (STAT3)/ nuclear factor -kappa B (NF-κB) pathway.

Chemoresistance in hepatocellular carcinoma (HCC) has been found to be influenced by exosomal transport of circRNAs. However, the role of circZFR in HCC chemoresistance still remains unclear. In the present study, circZFR was highly expressed in cisplatin (DDP)-resistant HCC cell lines and could regulate DDP resistance of the HCC cells. Also, circZFR was highly expressed in cancer-associated fibroblast (CAFs) and the exosome of CAFs. In addition, supplementation of CAFs in culture medium could promote DDP resistance of HCC cells. In vivo tumor xenograft experiments showed that knockdown of circZFR inhibited tumor growth and weakened DDP resistance, while CAFs-derived exosomes incubation increased the expression of circZFR, inhibited the STAT3/NF-κB pathway, promoted tumor growth, and enhanced DDP resistance. In general, CAFs-derived exosomes deliver circZFR to HCC cells, inhibit the STAT3/NF-κB pathway, and promote HCC development and chemoresistance. The results provided a new sight for the prevention and treatment of chemoresistance in HCC.

Metformin Suppresses Hepatocellular Carcinoma through Regulating Alternative Splicing of LGR4.

Methods: First, the expression of LGR4 in HCC tumor tissues and cell lines was detected by western blotting and immunofluorescence. The ability of cell proliferation, migration, and invasion was detected with CCK8, wound-healing, and transwell assays when overexpressing LGR4 or treating with metformin. The ß-catenin expression was detected by immunofluorescence. In order to investigate novel AS-associated LGR4, we discarded LGR4 isoforms from GSO databases. We used siRNA to knock down the specific isoform to check the cell proliferation, migration, and invasion when treated with metformin. Results: The level of LGR4 expression was higher in HCC cell lines and tumor tissues. The HCC cell proliferation, migration, and invasion were increased when overexpressing LGR4, which could be reduced by metformin treatment. The GEO database (GSE190076) showed that LGR4 had switching properties in HCC cell lines treated with metformin. We used siRNA to knock down the specific isoform, and the result showed that the specific isoform siRNA could promote the inhibition of cell invasion caused by metformin treatment. Conclusions: LGR4 could promote the ability of cell proliferation, migration, and invasion in HCC, which could be reduced by metformin through alternative splicing.

CircMYH9 increases KPNA2 mRNA stability to promote hepatocellular carcinoma progression in an EIF4A3-dependent manner.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed cancer worldwide with a high incidence of recurrence and metastasis; however, the molecular mechanisms underlying HCC development remain to be fully understood. In this study, we identified circMYH9 as an important regulator of HCC. Overexpression of circMYH9 induced, while knockdown of circMYH9 inhibited, the proliferation, migration, and invasion of HCC cells. Mechanistically, circMYH9 bound to eukaryotic translation initiation factor 4A3 (EIF4A3) and increased karyopherin subunit alpha 2 (KPNA2) mRNA stability. circMYH9 knockdown in HCC cells reduced the stability of KPNA2 mRNA. Importantly, circMYH9 regulation of HCC required the activity of KPNA2. In support with this, circMYH9 level was positively correlated with the expression of KPNA2 in HCC patient samples. Taken together, our study was the first to uncover the oncogenic role of circMYH9 in HCC and further elucidated the functional mechanism of circMYH9 by interacting with EIF4A3 to increase KPNA2 mRNA stability. Our findings might provide a novel potential target for the diagnose and treatment of HCC.

CircASPH Promotes Hepatocellular Carcinoma Progression Through Methylation and Expression of HAO2.

CircRNAs have been reported to be related to hepatocellular carcinoma (HCC) development. Limited studies have revealed the expression profile of circRNAs in tumor and para-tumor normal samples in HCC patients. We found that circASPH was significantly increased in HCC tumor samples and that the level of circASPH was closely related to the overall survival of HCC patients. Mechanistically, circASPH could regulate the methylation of the promoter and expression of hydrocyanic oxidase 2 (HAO2) to promote HCC progression by acting as a sponge for miR-370-3p, and miR-370-3p could target DNMT3b and increase the 5mC level. In summary, our study determined that circASPH could regulate the methylation and expression of HAO2 and it could be considered an important epigenetic regulator in HCC progression.

Multiscale Simulation on Product Distribution from Pyrolysis of Styrene-Butadiene Rubber.

Pyrolysis of styrene-butadiene rubber receives renewed attention due to its application in tackling the waste tire disposal problem while allowing energy recovery. The density functional theory calculation (DFT) and ReaxFF molecular dynamics simulation (MD) are adopted to study the pyrolysis process with the variation of temperature and pressure. The bond dissociation energies of intramonomer and intermonomer bonds in trimers with different linking methods are calculated by DFT, where the bond with low energy tends to break during the pyrolysis process. The following MD simulation shows the pyrolysis product distribution of chain segments in styrene-butadiene rubber, where bond breaking positions in MD agree well with corresponding results in DFT and experiment. The next nearest neighbor bonds (single bonds) connected with double bond or benzene usually have lower dissociation energies than other single bonds and prone to break during the pyrolysis process. And thus, the intermonomer bonds tend to break at relatively low temperatures (around 650 K in experiment) prior to intramonomer bonds, which result in the emergence of monomers. With the temperature increase, intramonomer bonds are broken and thus large fragments are further pyrolyzed into small ones (e.g., C2 and C). Besides, the pressure strongly influences the product distribution, where high pressures promote the occurrence of secondary reactions.

The long non-coding RNA Linc-GALH promotes hepatocellular carcinoma metastasis via epigenetically regulating Gankyrin.

Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer, and it is characterized by high rate of metastasis and recurrence. Recent studies have boosted our understanding that Gankyrin contributes to both of these pathological properties, but the mechanisms underlying its aberrant regulation are poorly understood. Recently, many long noncoding RNAs (lncRNAs) have been reported to be involved in regulating the expression of oncogenes and anti-oncogenes through various mechanisms. Here, using transcriptome microarray analysis, we identified a long intergenic noncoding RNA termed Linc-GALH that was highly expressed and concordance with Gankyrin expression in HCC. In addition, we revealed that Linc-GALH was an independent unfavorable prognostic indicator for HCC, followed functional experiments showed that Linc-GALH promoted HCC cells migration and invasion in vitro, and enhanced lung metastasis ability of HCC cells in vivo. Mechanistically, we found that Linc-GALH could regulate the expression of Gankyrin through controlling the methylation status of Gankyrin by adjusting the ubiquitination status of DNMT1 in HCC. Collectively, our results demonstrated the role and functional mechanism of Linc-GALH in HCC, and indicated that Linc-GALH may act as a prognostic biomarker and potential therapeutic target for HCC.

14-3-3ζ delivered by hepatocellular carcinoma-derived exosomes impaired anti-tumor function of tumor-infiltrating T lymphocytes.

Increasing evidence shows that the anti-tumor functions of tumor-infiltrating T lymphocytes (TILs) were inhibited significantly, but the underlying mechanisms remain not fully understood. In this study, we found that 14-3-3ζ expression was up-regulated in hepatocellular carcinoma (HCC) cells and in TILs. TILs with 14-3-3ζ high-expression (14-3-3ζhigh) exhibited impaired activation (CD69), proliferation (Ki67) and anti-tumor functions compared to 14-3-3ζ low expression (14-3-3ζlow) TILs. Flow cytometry assay showed that compared with 14-3-3ζlow CD8+T cells, 14-3-3ζhigh ones exhibited higher frequency of exhausted phenotypes as measured by inhibitory receptors such as PD-1, TIM-3, LAG3, and CTLA-4. 14-3-3ζ overexpression inhibited the activity and proliferation of peripheral blood CD3+ T cells, deviated the differentiation of naive T cells from effector T cells to regulatory T cells. Moreover, we found that 14-3-3ζ expression levels in TILs correlated positively with those in HCC cells. Naive T cells co-cultured with HCC cells or the visible components of culture medium of HCC cells exhibited increased 14-3-3ζ expression. Stochastic optical reconstruction microscopy (STORM) and confocal assay showed that 14-3-3ζ-containing exosomes derived from HCC cells could be swallowed by T cells, suggesting that 14-3-3ζ might be transmitted from HCC cells to TILs at least partially through exosomes. In conclusion, our study for the first time demonstrated that 14-3-3ζ is up-regulated in and inhibited the anti-tumor functions of tumor-infiltrating T cells in HCC microenvironment and that 14-3-3ζ might be transmitted from HCC cells to T cells at least partially through exosomes.

[Metabolism of mitomycin C by human liver microsomes in vitro].

To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4. 0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10. 0, 14. 0, 14. 8 min ( named as Ml, M2, M3) , respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0. 52 (95% CI, 0. 40 - 0.67) mmol x L(-1), 0. 81 (95% CI, 0. 59 - 1. 10) mmol x L(-1), 0. 54 (95% CI, 0. 41 -0. 71) mmol x L(-1) , respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it' s dose-effect fitted to Sigmoidal curve and it' s IC50 was 59. 68 (95% CI, 40. 66 - 87. 61) micromol x L(-1) , which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.