Mechanistic Study Of Monocrotaline-and Diosbulbin B-induced Hepatotoxicity

Pyrrolizidine alkaloids(PAs)are widespread hepatotoxins,occur in varieties of plant species.Exposure to PAs may cause severe hepatic injury,pulmonary toxicity,carcinogenesis,and genotoxicity.Numerous PAs poisoning cases have been documented in many countries.PAs have become a potential public health issue to humans and livestock.Monocrotaline is a retronecine-type PAs found in the plant family leguminosae(genus Crotalaria).Monocrotaline was selected to explore the mechanisms of PAs-induced hepatotoxicitiesFuran-containing compounds refer to a class of compounds containing furan ring(s),which are abundant in fruits,herbs,foods,and beverages.Many furan-containing compounds have been reported to be toxicities.The risks for intake of toxic furans have been rising,due to the rapid growth of globe-wide consumption of medical remedies,dietary supplements,and"natural" foods.Diosbulbin B(DIOB),a furanoid,is a major constituent of herbal medicine Dioscorea bulbifera L.Exposure to DIOB caused liver injury in humans and experimental animals.The mechanisms of DIOB-induced hepatotoxicities remain unknown.The studies performed are summarized as below.1.The execution of the toxicities of the alkaloids requires metabolic activation.Protein modificcation by reactive metabolites of PAs has been suggested to be an important mechanism of the toxic actions of PAs.Studies showed that dehydro-PAs modified cysteine residues of hepatic proteins in animals administered with PAs.The objectives of the present study were to define the interactions of dehydromonocrotaline(DHM)with lysine,lysine derivatives.The reaction of DHM with L-lysine produced three DHM-derived adducts,namely A1’-A3’,The observed molecular ion of A1’(m/z 410),along with its mass spectrum,indicates that the adduct contained two molecules of Lys,named as 7,9-di-Lys-DHP.The adducts A2’ and A3’observed were most likely the two regioisomers resulting from alkylating at C7 and C9 of the necine base,i.e.7-Lys-DHP and 9-Lys-DHP respectively.7-Lys-DHP was the major mono-Lys-DHP adduct.A lysine-containing peptide(8 amino acids)was employed for the investigation of the interaction of Lys residues with DHM.The observation of b3*and y6*,along with the retaining of b2 and y5,indicates the modification of Lys residue of the peptide by the necine base.A model protein,bovine serum albumin was used to explore the lysine modification of proteins by DHM.DHM was found to react with ε-amino group of all model compounds tested after incubation with DHM,and the modification reaction preferentially occurred at C7 of the necine base.The lysine residue modification with the same regioselectivity was also observed in hepatic proteins of mice treated with monocrotaline.The observed modification increased with the increasing in the doses administered in the animals.This work allowed us to better understand the mechanisms of hepatotoxicity of monocrotaline.2.Dehydro-PAs are well-known bidentate alkylating agents with two electrophilic benzylic-like carbons at C7 and C9,thus it can react with DNA and protein to form DNA-protein cross-links.DPCs strongly disrupt normal DNA-protein interactions and interfere with DNA replication,transcription,and repair,which ultimately threatens genomic integrity and cell viability.DHM can react with lysine/cysteine,dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine),respectively.As an initial step,we incubated DHM with lysine/cysteine and dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine),and cross-link adducts were monitored by the AB Sciex 4000 Q-Trap MS.A dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine)containing DNA(ctDNA)was employed for the investigation of the interaction of DHM with dG/dA and lysine/cysteine,the cross-linked DNA samples were enzymatically hydrolyzed to nucleosides with DNAase I,phosphodiesterase I,and alkaloid Phosphatase.Five analytes showed identical chromatographic and mass spectrometric behaviors as those of authentic Cys-DHP-dG adducts were detected in the mixture,and no such adduct was found in the digestion mixture without DHM.A lysine/cysteine containing protein(BSA)was employed for the investigation of the interaction of DHM with dG/dA and lysine/cysteine,the cross-linked protein samples were enzymatically hydrolyzed to amino acids with α-chymotrypsin and pronase E.Two Cys-DHP-dG adducts were detected in the mixture,and no such adduct was found in the digestion mixture without DHM.In addition,DHM reacts with BSA and ctDNA to form DNA-protein cross-links in vitro.Only 7(R)-Cys-DHP-dG was observed in hepatic homogenates of mice treated with monocrotaline.This work allowed us to better understand the mechanisms of hepatotumorigenicity of monocrotaline.3.The hepatotoxicities of DIOB were evaluated in detail.Intraperitoneal administration of DIOB caused elevations of serum ALT activities in a time-dependent and dose-dependent manner.A single dose of 200 mg/kg produced over twenty-fold elevations in serum ALT activities.Histopathologic evaluation showed focal necrosis in the liver of mice given DIOB at 200 mg/kg.Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB,increased area under the concentration-time curve of blood DIOB,decreased urinary excretion of GSH conjugates derived from DIOB,and increased urinary excretion of parent drug.Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities.In order to define the role of furan moiety in DIOB-induced liver toxicities,we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB.No liver injury was observed in the animals given the same doses of tetrahydro-DIOB.The furan moiety was essential for DIOB-induced hepatotoxicities.The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities.The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites,and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules.Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.