Trends in deaths and disability-adjusted life-years of stroke attributable to high body-mass index worldwide, 1990-2019.

Background: High body mass index (HBMI) is an independent risk factor for stroke. Previous studies on the incremental burden of the rapid growth of stroke attributable to HBMI are incomplete and lag behind. We aim to assess the global burden of stroke attributable to HBMI based on a public database online. Materials and methods: Study data were taken from the Global Burden of Disease, Injuries, and Risk Factors Study; deaths, the Disability-Adjusted Life-Years (DALYs), and their age-standardized rates were screened. The join point regression was used, wherein age-standardized rates were referred to as temporal trends in disease burden. Results: Deaths from stroke attributable to HBMI worldwide were on the rise during 1990-2019, with an increase of 88.75%. Age-standardized DALYs were on the rise during 1990-2003 but declined during 2003-2013, with a turning point in 2013 and an increasing trend since then [the Annual Percentage Change (APC) = 0.30%, p < 0.05]. China, India, Indonesia, the Russian Federation, and the United States of America shared in sequence the rate of leading deaths and DALYs in 2019. The Socio-Demographic Index (SDI) was associated with an increasing trend in age-standardized deaths (R = -0.24, p < 0.001) and age-standardized DALYs (R = -0.22, p = 0.0018). Conclusion: A range of indicators for the global burden of stroke attributable to HBMI have been on the rise for the past three decades. Tremendous efforts worldwide should be in place to control and treat stroke attributable to HBMI, especially in regions with high-middle and middle SDIs and among middle-aged and aged populations.

Glutathione Conjugation and Protein Adduction by Environmental Pollutant 2,4-Dichlorophenol In Vitro and In Vivo.

2,4-Dichlorophenol (2,4-DCP), an environmental pollutant, was reported to cause hepatotoxicity. The biochemical mechanisms of 2,4-DCP induced liver injury remain unknown. The present study showed that 2,4-DCP is chemically reactive and spontaneously reacts with GSH and bovine serum albumin to form GSH conjugates and BSA adducts. The observed conjugation/adduction apparently involved the addition of GSH and departure of chloride via the ipso substitution pathway. Two biliary GSH conjugates and one urinary N-acetyl cysteine conjugate were observed in rats given 2,4-DCP. The N-acetyl cysteine conjugate was chemically synthesized and characterized by mass spectrometry and NMR. As expected, 2,4-DCP was found to modify hepatic protein at cysteine residues in vivo by the same chemistry. The observed protein adduction reached its peak at 15 min and revealed dose dependency. The new findings allowed us to better understand the mechanisms of the toxic action of 2,4-DCP.

Metabolic Activation of Tofacitinib Mediated by Myeloperoxidase in Vitro.

Tofacitinib (TFT) is used for the treatment of moderately and severely active rheumatoid arthritis. Unfortunately, TFT was reported to induce leukopenia, and the underlying mechanisms remain unclear. The present study demonstrated that TFT was oxidized to a chemically reactive nitrenium ion by myeloperoxidase (MPO) occurring in neutrophils. The electrophilic ion showed chemical reactivity toward N-acetyl-cysteine (NAC) to produce two TFT-NAC conjugates (M1 and M2) in incubation of TFT with leucocytes in the presence of NAC. The generation of the nitrenium ion was verified by HClO-mediated oxidation of TFT. In addition, the nitrenium ion was found to react with sulfhydryl groups of cysteine residues of cellular protein in leucocytes after exposure to TFT. The study facilitates the understanding of the mechanisms of TFT toxic action.

Tofacitinib Is a Mechanism-Based Inactivator of Cytochrome P450 3A4.

Tofacitinib (TFT) is an oral JAK inhibitor which has been approved for the treatment of moderately and severely active rheumatoid arthritis. TFT was found to show concentration-, time-, and NADPH-dependent inhibition of CYP3A4, and irreversibility of the inactivation was also observed. Incubation (40 min, 37 °C) of recombinant CYP3A4 with TFT at 200 µM resulted in >70% loss of CYP3A4 activity. Estimated kinact and KI were 0.037 min-1 and 93.2 µM, respectively. GSH and superoxide dismutase/catalase revealed minor or little protection against the CYP3A4 inactivation. Furthermore, ketoconazole attenuated TFT-mediated CYP3A4 inactivation. Epoxide and α-keto-aldehyde intermediates of TFT were trapped and characterized in microsomal incubations, respectively. The aldehyde intermediate is believed to be the key for the enzyme inactivation. Multiple P450 enzymes, including CYPs2C19, 3A4, 2D6, and 1A2, participated in the metabolism of TFT to the epoxide, while the formation of the aldehyde was mainly catalyzed by CYP3A4. In conclusion, TFT was proven to be a mechanism-based inactivator of CYP3A4.

Studies on hepatotoxicity and toxicokinetics of colchicine.

Colchicine (COL) is an alkaloid existing in plants of Liliaceous colchicum. It has widely been used in the treatments of many diseases, such as gout, Familial Mediterranean Fever, and tumor. However, the adverse effects of COL are an obstacle to its safe use. The present studies explored the role of metabolic demethylation in the development of COL-induced hepatotoxicity. We found that inhibition of CYP3A increased the susceptibility of mice to COL hepatotoxicity, and induction of CYP3A decreased the susceptibility of animals to the hepatotoxicity. The toxicokinetic study demonstrated that pretreatment with ketoconazole caused elevated area under the concentration-time curve of COL. Three demethylation metabolites of COL were found to be less hepatotoxic than the parent compound. It appears that the formation of electrophilic demethylation metabolites was not involved in the development of COL-induced liver injury.

Metabolic Activation and Cytotoxicity of Aloe-Emodin Mediated by Sulfotransferases.

Aloe-emodin (AE) is a major anthraquinone ingredient of numerous traditional Chinese medicines with a variety of beneficial biological activities in vitro. Previous studies suggested that AE possessed cytotoxicity and genotoxicity. Nevertheless, the mechanisms of the toxic action of AE have not yet been fully clarified. The present study aimed at characterization of metabolic pathways of AE to better understand the mechanisms of AE-induced cytotoxicity. An AE-derived glutathione conjugate (AE-GSH) was observed in rat liver cytosol incubations containing AE and GSH, along with 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Similar incubation fortified with N-acetylcysteine (NAC) in place of GSH offered an AE-NAC conjugate corresponding to the GSH conjugate. The formation of the two conjugates was found to require PAPS. The two conjugates were respectively detected in bile and urine of rats given AE. Sulfotransferase (SULT) inhibitor pentachlorophenol (PCP) suppressed the production of the observed AE-GSH/NAC conjugates in vivo, which suggested that SULTs participated in the process of the metabolic activation of AE. The presence of PCP attenuated cell susceptibility to AE-induced cytotoxicity. The present study illustrated potential association of sulfation-mediated bioactivation of AE with its cytotoxicity.

In vitro and in vivo studies of the metabolic activation of chelidonine.

Chelidonium majus L. is a herbal medicine widely employed in Europe and Western Asia. Chelidonine (CHE) is a major constituent of the herb and has been reported to be an inhibitor of the cytochrome P450 enzymes (CYP). The major objective of the present study was to study the metabolic pathways of CHE in order to identify potential reactive metabolites responsible for the enzyme inhibition. Three oxidative metabolites (M1-M3) were detected in human liver microsomal incubations after exposure to CHE. M1 and M2 were two isomers of catechol derivatives, and M3 was a dicatechol compound. The M1-M3 metabolites were also observed in bile of rats given CHE. A total of five glutathione (GSH) conjugates (M4-M8) were detected in microsomes containing CHE, GSH, and NADPH. Moreover, M4 and M6 originated from M1, M5 and M7 resulted from M2, and M8 was a M3-derived GSH conjugate. Three biliary CHE-derived GSH conjugates (M4, M5 and M8) were found in CHE-treated rats. This indicates that CHE was bioactivated to ortho-quinone derivatives both in vitro and in vivo. Recombinant P450 enzyme incubations demonstrated that the CYPs3A4, 1A2, 2C19 and 2D6 were mainly involved in metabolic activation of CHE. This study generated data that may be useful in understanding possible mechanisms of CHE-induced P450 inhibition.

Identification of Quinone Methide Intermediate Resulting from Metabolic Activation of Icaritin in Vitro and in Vivo.

Many herbal medicines such as epimedium have been reported to cause adverse effects, and icaritin is the common aglycone of many glucosides in epimedium. Our present work aimed at the clarification of the metabolic activation of icaritin possibly responsible for the adverse effects of epimedium. A quinone methide metabolite (M1) was detected in icaritin-fortified microsomal incubations. A glutathione (GSH) conjugate (M2) and N-acetyl-l-cysteine (NAC) conjugate (M3) derived from icaritin were observed in GSH/NAC-supplemented rat/human liver microsomal incubations. CYP3A family was the predominant enzyme catalyzing the bioactivation of icaritin. In conclusion, sufficient evidence indicates the metabolic activation of icaritin to quinone methide metabolite.

Metabolism-based herb-drug interaction of Corydalis Bungeanae Herba with berberine in vitro and in vivo in rats.

Corynoline (CRL) and berberine (BER) are the major bioactive components found in traditional Chinese medicines Corydalis Bungeanae Herba (Corydalis bungeanae) and Coptidis Rhizoma, respectively. The two herbs serve as anti-inflammatory agents and are generally applied to many prescriptions. The aims of the study were to evaluate herb-drug interaction of C. bungeanae with BER and to investigate the mechanisms of the interaction action. Pre-treatment of BER caused reduction of plasma CRL in rats with increased formation of its three oxidative metabolites (M1-M3). Compared with the vehicle-treated group, the peak concentration and area under the concentration-time curve of CRL decreased by ~60% (given CRL) and ~50% (given extracts) in rats pre-treated with BER, respectively, along with 130 and 100% increases in apparent clearance. More M1-M3 were formed in liver microsomes of rats pretreated with BER (7 days) than in those pretreated with vehicle. Additionally, elevated activities of rCYPs2D2 and 1A2 (CYPs2D6 and 1A2) were observed in the BER-induced group. Up-regulated expression of hepatic rCYP2D2 (CYP2D6) was found in animals after 7 days of treatment of BER. The study illustrated that C. Bungeanae and BER produced metabolic herb-drug interaction and provided important information that combination of C. bungeanae with BER-containing herbal medicines may encounter the risk of decreased efficacy of CRL.

A Difference in Internal Exposure Makes Newly Weaned Mice More Susceptible to the Hepatotoxicity of Retrorsine Than Adult Mice.

Pyrrolizidine alkaloids (PAs) are known hepatotoxins. Children have been reported to be particularly susceptible to PA-induced hepatotoxicity. To improve our understanding of the mechanisms of the age-dependent difference in susceptibility to PA hepatotoxicity, a comparative study of hepatotoxicities of retrorsine (RTS), a representative PA, was performed in newly weaned (3-4 weeks of age) and adult mice (8-12 weeks of age). Intraperitoneal administration of RTS at a dose of 50 mg/kg induced limited increases in serum ALT and AST activities in adult mice, while the same dosage of RTS caused intensified increases in serum ALT and AST activities in newly weaned mice. Toxicokinetic and metabolic activation studies in vitro and in vivo were performed to examine the factors responsible for the observed difference in toxicity susceptibility in mice of both ages. The values of AUC0-4h in plasma and liver of newly weaned mice are higher (all by 1.4-fold) than those in adult mice given the same dosage of RTS. As expected, more plasma pyrrolic ester-glutathione (GSH) conjugates (1.3-fold more) and pyrrolic ester-derived hepatic protein adduction (1.3-fold more) were found in newly weaned mice. Administration of RTS induced dramatic decreases in hepatic GSH levels (as little as 55% remained) of newly weaned mice, while the same dose of RTS did not reveal such GSH depletion in adult mice. The Vmax/ Km (CLint) for RTS metabolic activation in newly weaned mouse liver microsomes was found to be similar to that of adult mice. In conclusion, more internal exposure of RTS made newly weaned mice more susceptible to PA-induced hepatotoxicity.

Metabolite profile analysis and pharmacokinetic study of emodin, baicalin and geniposide in rats.

1. Emodin, baicalin and geniposide are the major bioactive components in Da-Huang, Huang-Qin and Zhi-Zi which are herbal medicines widely used in Asian nations. 2. The metabolism of the three compounds was found to undergo hydroxylation, decarboxylation, dehydration, methylation, hydrolysis, hydrogenation, dihydrogenation, sulfation, glucosidation and/or glucuronidation. A total of 63 metabolites were detected in urine, plasma and bile of rats given a mixture of the three compounds. 3. Pharmacokinetic properties of the three compounds were determined in rats given the extracts of Da-Huang, Huang-Qin and Zhi-Zi. The pharmacokinetic parameters for emodin, baicalin and geniposide were found to be 0.13 ± 0.11, 0.25 ± 0.12 and 0.40 ± 0.09 h (Tmax); 21 ± 9, 1515 ± 254 and 482 ± 50 ng/mL (Cmax); 8.6 ± 5.5, 18.3 ± 2.8 and 22.1 ± 17.2 h (t1/2); 29 ± 20, 16886 ± 3734 and 2936 ± 551 ng/mLch (AUC(0-t)); and 37 ± 20, 22624 ± 6295 and 3582 ± 820 ng/mLch (AUC(0-∞)). 4. The metabolism and pharmacokinetic studies facilitate appropriate employment of Da-Huang, Huang-Qin and Zhi-Zi in clinic.

Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo.

Diosbulbin B (DIOB), a hepatotoxic furan-containing compound, is a primary ingredient in Dioscorea bulbifera L., a common herbal medicine. Metabolic activation is required for DIOB-induced liver injury. Protein covalent binding of an electrophilic reactive intermediate of DIOB is considered to be one of the key mechanisms of cytotoxicity. A bromine-based analytical technique was developed to characterize the chemical identity of interaction of protein with reactive intermediate of DIOB. Cysteine (Cys) and lysine (Lys) residues were found to react with the reactive intermediate to form three types of protein modification, including Cys adduction, Schiff's base, and Cys/Lys crosslink. The crosslink showed time- and dose-dependence in animals given DIOB. Ketoconazole pretreatment decreased the formation of the crosslink derived from DIOB, whereas pretreatment with dexamethasone or buthionine sulfoximine increased such protein modification. These data revealed that the levels of hepatic protein adductions were proportional to the severity of hepatotoxicity of DIOB.

Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids Retrorsine and Monocrotaline.

Many pyrrolizidine alkaloids (PAs) can cause liver injury in animals and humans. Different hepatotoxic PAs can produce similar hepatotoxic effects, but the degree of their toxicities may vary widely. Retrorsine (RTS) and monocrotaline (MCT) share the same core structure (retronecine) and similar metabolic activation pathway. RTS and MCT both produced liver injury, but the former was more hepatotoxic than the latter. Enzyme kinetic study demonstrated that the value of Vmax/Km for RTS was 5.5-fold larger than that of MCT. Additionally, RTS produced higher levels of pyrrole-glutathione (GSH) conjugates and protein covalent binding than MCT at the same dose. Furthermore, RTS induced significant hepatic GSH depletion but MCT did little. This comparative study provides clear evidence that the generation of the reactive pyrrolic intermediates plays a critical role in PA-induced hepatotoxicity.

Cysteine-Based Protein Adduction by Epoxide-Derived Metabolite(s) of Benzbromarone.

Benzbromarone (BBR) is a therapeutically useful uricosuric agent but can also cause acute liver injury. The hepatotoxicity of BBR is suggested to be associated with its metabolic activation. Our recent metabolic study demonstrated that BBR was metabolized to epoxide intermediate(s) by cytochrome P450 3A, and the intermediate(s) was reactive to N-acetylcysteine. The objectives of the present study were to determine the chemical identity of the interaction of protein with the epoxide intermediate(s) of BBR and to define the association of the protein modification with hepatotoxicity induced by BBR. Microsomal incubation study showed that the reactive intermediate(s) covalently modified microsomal protein at cysteine residues. Such adduction was also observed in hepatic protein obtained from liver of mice given BBR. The protein covalent binding occurred in time- and dose-dependent manners. Pretreatment with ketoconazole attenuated BBR-induced protein modification and hepatotoxicity, while pretreatment with dexamethasone or buthionine sulfoximine potentiated the protein adduction and hepatotoxicity induced by BBR. A good correlation was observed between BBR-induced hepatotoxicity and the epoxide-derived hepatic protein modification in mice. The present study provided in-depth mechanistic insight into BBR-induced hepatotoxicity.

Lysine- and cysteine-based protein adductions derived from toxic metabolites of 8-epidiosbulbin E acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional herbal medicine widely used in Asian nations. Our early studies demonstrated that administration of EEA caused acute hepatotoxicity in mice and the observed toxicity required P450-mediated metabolic activation. Protein modification by reactive metabolites of EEA has been suggested to be an important mechanism of EEA-induced hepatotoxicity. The objectives of the present study were to investigate the interaction of the electrophilic reactive metabolites derived from EEA with lysine and cysteine residues of proteins and to define the correlation of protein adductions of EEA and the hepatotoxicity induced by EEA. EEA-derived cis-enedial was found to modify both lysine and cysteine residues of proteins. The observed modifications increased with the increase in doses administered in the animals. The formation of protein adductions derived from the reactive metabolites of EEA were potentiated by buthionine sulfoximine, but were attenuated by ketoconazole. This work facilitated better understanding of the mechanisms of toxic action of EEA.

Simultaneous assessment of endogenous thiol compounds by LC-MS/MS.

Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC-MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC-MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1-400µM. The lowest limits of detection (S/N=3) in a range from 0.31fmol (for NAC) to 4.98fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.

Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ.

Lactoferrin-modified PEGylated liposomes loaded with doxorubicin for targeting delivery to hepatocellular carcinoma.

Lactoferrin (Lf) is a potential-targeting ligand for hepatocellular carcinoma (HCC) cells because of its specific binding with asialoglycoprotein receptor (ASGPR). In this present work, a doxorubicin (DOX)-loaded, Lf-modified, polyethylene glycol (PEG)ylated liposome (Lf-PLS) system was developed, and its targeting effect and antitumor efficacy to HCC was also explored. The DOX-loaded Lf-PLS system had spherical or oval vesicles, with mean particle size approximately 100 nm, and had an encapsulation efficiency of 97%. The confocal microscopy and flow cytometry indicated that the cellular uptake of Lf-PLS was significantly higher than that of PEGylated liposome (PLS) in ASGPR-positive cells (P<0.05) but not in ASGPR-negative cells (P>0.05). Cytotoxicity assay by MTT demonstrated that DOX-loaded Lf-PLS showed significantly stronger antiproliferative effects on ASGPR-positive HCC cells than did PLS without the Lf modification (P<0.05). The in vivo antitumor studies on male BALB/c nude mice bearing HepG2 xenografts demonstrated that DOX-loaded Lf-PLS had significantly stronger antitumor efficacy compared with PLS (P<0.05) and free DOX (P<0.05). All these results demonstrated that a DOX-loaded Lf-PLS might have great potential application for HCC-targeting therapy.

In vitro and in vivo studies of the metabolic activation of 8-epidiosbulbin E acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our preliminary studies demonstrated that administration of EEA caused acute hepatotoxicity in mice, and the observed toxicity required cytochromes P450-mediated metabolism. Metabolic activation studies of EEA were performed in vitro and in vivo. Microsomal incubations of EEA supplemented with N-acetyl lysine (NAL) and glutathione (GSH) generated six metabolites (M1-M6). M1-M4 were characterized as pyrrole derivatives, and M5 and M6 were pyrrolinones. M2-M6 were detected in bile and/or urine of rats given EEA. Dimethyldioxirane-mediated oxidation of EEA in the presence of NAL and GSH produced M1-M6, all of which were generated in microsomal incubations. The structures of M3 and M6 were confirmed by (1)H and (13)C NMR. These findings provide evidence for the metabolic activation of EEA to the corresponding cis-enedial intermediate both in vitro and in vivo. Ketoconazole inhibited the microsomal production of the cis-enedial, and P450 3A4 was found to be the primary enzyme involved in the bioactivation of EEA.