Txawv lub raum Chronotoxicity Ntawm Bromobenzene Thiab Nws Cov Metabolites Intermediate hauv nas

Bromobenzene (BB) paub tias ua rau muaj kev hem thawj rau tib neeg kev noj qab haus huv. Peb yav dhau los qhia tau hais tias BB tau qhia chronotoxicity, uas yog, kev hloov pauv txhua hnub hauv qhov hnyav ntawm hepatotoxicity tshwm sim hauv cov nas. Txawm hais tias BB tau pom tias muaj nephrotoxicity me me, qhov hloov pauv txhua hnub tsis tau pom nyob rau hauv qhov tshuaj lom no. Qhov no yuav raug ntaus nqi rau qhov tseeb tias BB-induced chronotoxicity tsuas yog pom hauv daim siab thiab tsis nyob hauvlub raumthiab tias qhov kev puas tsuaj los ntawm BB yog qhov tseem ceeb hauv daim siab, npog qhov hloov pauv txhua hnub hauv nephrotoxicity. Txhawm rau kom paub tseeb tias ob qho no, peb tau tshuaj xyuas qhov hloov pauv txhua hnub hauv nephrotoxicity vim yog BB intermediate metabolites uas tsom raulub raum: 3-bromophenol, bromohydroquinone, thiab 4-bromocatechol. Cov nas tau txhaj nrog 3-bromophenol, bromohydroquinone, lossis 4-bromocatechol intraperitoneally ntawm rau lub sijhawm sib txawv hauv ib hnub (zeitgeber time (ZT): ZT2, ZT6, ZT10, ZT14, ZT18, lossis ZT22). Kev tuag tau saib xyuas rau 7 d tom qab txhaj tshuaj. Cov nas tau nkag siab ntau dua rau qhov mob toxicity ntawm cov metabolites nyob ib ncig ntawm ZT14 (dub-theem) raug ntau dua li ntawm ZT2 (lub teeb-theem) raug. Tsis tas li ntawd, cov nas uas tau tswj hwm nrog cov koob tshuaj uas tsis ua rau tuag taus ntawm 4-bromocatechol pom tias muaj qhov nce ntxiv hauv cov ntshav plasma urea nitrogen thiablub raumMalondialdehyde ntawm ZT14 raug. Ntxiv mus, glutathione peroxidase-4, qhov taw qhia ferroptosis, tau txo qis ntawm ZT14 raug. Cov txiaj ntsig no qhia tau hais tias muaj kev phom sij ntawm BB metabolites siab dua thaum lub sijhawm tsaus ntuj tshwm sim, thiab ua kom pom tias yog vim li cas vim li cas qhov kev hloov pauv ntawm nephrotoxicity los ntawm BB tsis tau pom nyob rau hauv peb tsab ntawv tshaj tawm dhau los.lub raumkev puas tsuajtau npog vim mob siab heev.

Nyem rau Cistanche tubulosa ntsiav tshuaj rau mob raum

Cov ntsiab lus bromobenzene; variation ntawm diurnal; circadian atherosclerosis; chronotoxicity;raum

Taw qhia

Bromobenzene (BB) yog ib qho kev lag luam reagent uas feem ntau yog siv los ua cov khoom siv raw rau kev sib txuas ntawm cov tshuaj, tshuaj zawv plaub hau, thiab tshuaj tua kab.1,2) BB yog cov kuab tshuaj organic uas ua rau lub ntuj raug txim thiab ua rau muaj kev hem thawj rau tib neeg kev noj qab haus huv. . Ib txoj kev tshawb fawb tau hais tias BB ua rau muaj mob hepatotoxicity hnyav thiab mob melub raumtoxicity nyob rau hauv nas.3) Txawm hais tias BB nws tus kheej, ib daim ntawv uas tsis yog-metabolite, muaj toxicity tsawg, nws intermediate metabolites muaj tshuaj lom rau lub siab thiablub raum. Hepatic CYP450s, xws li CYP1A2, CYP1B1, thiab CYP2E1, metabolize BB rau plaub metabolites: 3-bromophenol (3-BrP), bromohydroquinone (BrHQ), 4-bromocatechol (4-} BrC), thiab BB-3,4-oxide. BB-3,4-Oxide nthuav tawm siab hepatotoxicity thiab lwm cov metabolites qhia nephrotoxicity,4–6) qhia tias cov metabolites muaj lub hom phiaj sib txawv.

Hauv txoj kev tshawb no, peb tau tsom mus rau "chronotoxicology" raws li peb tau hais yav dhau los txog kev sib raug zoo ntawm lub sijhawm tswj hwm thiab cov tshuaj lom neeg hnyav.7–9) Peb txoj kev tshawb fawb yav dhau los tau pom muaj kev hloov pauv tseem ceeb hauv txhua hnub hauv qhov hnyav ntawm cov tshuaj lom BB hauv cov nas, piv txwv li. , nas tau ua siab ntev rau BB-induced hepatic raug mob thaum lub sij hawm tsaus ntuj-theem raug (2:00) dua li thaum lub teeb-theem raug (14:00).8) Txawm hais tias BB pom me ntsis nephrotoxicity , chronotoxicity tsis tau pom nyob rau hauvlub raum. Yog li, BB-induced chronotoxicity tau pom tsuas yog hauv daim siab thiab tsis nyob hauvlub raum. Hauv peb txoj kev tshawb fawb yav dhau los, streptomycin, tshuaj tua kab mob uas paub tias yuav ua rau muaj kev cuam tshuam nephrotoxicity, qhia meej diurnal variation nyob rau hauv induced nephrotoxicity nyob rau hauv nas. tuslub raum, yog li npog lub chronotoxicity hauv lub cev tom kawg. Cov metabolites ntawm BB generated los ntawm ntau yam metabolic txoj kev tshwm sim nyob rau hauv tsawg concentrations thiab yog li yuav pab tau rau negligible kev puas tsuaj rau lub raum; Vim qhov no, BB metabolite-induced nephrotoxicity tsis tuaj yeem kwv yees li kev kho mob. Hauv txoj kev tshawb no, peb tau tshuaj xyuas qhov txawv ntawm diurnal nyob rau hauv nephrotoxicity ntawm BB intermediate metabolites: 3-BrP, BrHQ, thiab 4-BrC (Fig. 1).

Cov khoom siv thiab cov txheej txheem

Kev kho tsiaj

Txiv neej ICR nas tau yuav los ntawm Nyiv SLC Inc. (Shizuoka, Nyiv) thiab tau khaws cia nyob rau hauv cov txheej txheem ntawm kev tswj kub (24 ± 1 degree), av noo (55 ± 5%), thiab lub teeb (12: 12-h Lub teeb / tsaus nti, teeb rau ntawm 08: 00), nrog rau kev nkag mus rau dej thiab zaub mov dawb. Kev sim tshuaj tau ua los ntawm kev siv 7-cov tsiaj hnub nyoog ib lim piam. Tom qab ua tiav qhov kev sim, cov nas uas muaj sia nyob tau muab txi rau siv pentobarbital. Txhua qhov kev sim tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Kev Ua Haujlwm ntawm Kinjo Gakuin University.

Kev sim raws tu qauv

Rau qhov kev ntsuam xyuas kev tuag, {{0}}ib lub lis piam txiv neej ICR nas tau txais ib qho tshuaj intraperitoneal (ip) ntawm 500 mg/kg ntawm 3-BrP, 180 mg/kg ntawm BrHQ, lossis 220 mg / kg ntawm 4-BrC (ntawm qhov ntim ntawm 0.1 mL / 10 g ntawm lub cev hnyav; Tokyo Kasei Corp, Tokyo, Nyiv). Qhov koob tshuaj no yog txiav txim siab raws li tus nqi LD50 hauv cov ntaub ntawv kev nyab xeeb rau txhua yam tshuaj. Rau kev txhaj tshuaj ntawm txhua tus metabolite, nas tau muab rau rau pawg ntawm tsib tus tsiaj txhua. BB metabolites tau tswj hwm ntawm rau lub sijhawm sib txawv (lub sijhawm teev: 10:00, 14:00, 18:00, 22:00, 02: {{21 } }, thiab 06 : 00), piav raws li lub sij hawm zeitgeber (ZTs): ZT2, ZT6, ZT10, ZT14, ZT18, thiab ZT22, raws li. Cov nas tswj tau txais tib lub ntim ntawm saline. Kev tuag tau saib xyuas rau 7 d tom qab txhaj tshuaj.

Rau kev txheeb xyuas circadian variations hauvlub raumraug mob, 7-Ib hnub nyoog txiv neej ICR nas (5–9 nas) tau txhaj tshuaj ip ntawm 165 mg/kg ntawm 4-BrC ntawm ZT2 lossis ZT14. Cov nas tswj tau txais tib lub ntim ntawm saline raws li tau piav qhia saum toj no. Ani mals tau euthanized siv pentobarbital, thiab lawv cov plasma kuaj tau sau 24 teev tom qab txhaj tshuaj. Cov qauv plasma tau muab khaws cia ntawm -80 degree. Covlub raumntawm tag nrho cov tsiaj tau sau thiab khaws cia ntawm -80 degree.

Qib ntawm Plasma Ntshav Urea Nitrogen (BUN), Aspartate Aminotransferase (AST), thiab raum Malondialdehyde (MDA)

Plasma BUN thiab AST theem tau txiav txim siab siv BUN Wako Test thiab transaminase CII-Test Wako (FUJIFILM Wako Chemicals, Osaka, Nyiv), raws li cov chaw tsim khoom cov lus qhia.8) Tag nrholub raumMDA qib tau ntsuas los ntawm kev ntsuam xyuas colorimetric microplate (Oxford Biomedical Research, MI, USA), raws li cov chaw tsim khoom cov lus qhia.8)

Western Blot Analysis

Protein samples (50 µg) muab rho tawm los ntawmlub raumtau sib cais ntawm 10 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis.10) Cov tshuaj tiv thaiv thawj zaug tau siv raws li hauv qab no (tag nrho cov diluted 1: 1500): nas receptor-interacting protein (RIP) 1 monoclonal antibody, nas RIP3 monoclonal antibody, thiab nas glutathione peroxidase 4 (GPX4) monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); luav cleaved caspase-3 polyclonal antibody (Cell Signaling Technology); thiab nas -actin monoclonal antibody (Medical & Biological Laboratories Co., Ltd., Aichi, Nyiv).

Kev txheeb cais

Ntau qhov kev sib piv tau ua los ntawm Tukey-Kramer post-hoc xeem. Txhua qhov kev txheeb xyuas txheeb cais tau ua tiav siv SPSS 25.0 software (Chicago, IL, USA). Cov txiaj ntsig nrog p< 0.05="" were="" considered="" statistically="">

TSEEM CEEB THIAB PAUB

Hauv txoj kev tshawb no, peb piv cov chronotoxicity ntawm peb BB intermediate metabolites (3-BrP, BrHQ, thiab 4-BrC) uas paub tias yog induce.lub raumtshuaj lom. Peb thawj zaug tshawb xyuas qhov cuam tshuam ntawm lub sijhawm txhaj tshuaj ntawm BB intermediate metabolite-induced mortality. Daim duab 2 qhia txog cov tsiaj muaj sia nyob thiab lub sij hawm muaj sia nyob nruab nrab (MST) 7 d tom qab txhaj tshuaj. Thaum 3-BrP raug tswj hwm ntawm ZT10 thiab ZT14, txhua tus nas tuag (0 feem pua ​​) hauv 4 teev (Daim duab 2A). Hauv qhov sib piv, thaum 3-BrP tau tswj hwm ntawm ZT6 thiab ZT22, peb nas (60 feem pua) tau muaj sia nyob; thaum tswj hwm ntawm ZT2 thiab ZT18, tus naj npawb ntawm cov nas muaj sia nyob yog ib thiab ob (20 thiab 40 feem pua), feem. Ua raws li kev tswj hwm BrHQ, kev tuag ntawm nas tau pom tsuas yog hauv 4 teev tom qab kev tswj hwm hauv txhua pawg ZT. Thaum 7 d tom qab kev tswj hwm, tsis muaj nas muaj sia nyob hauv pawg ZT14 thiab ZT18 thiab peb (60 feem pua) muaj sia nyob hauv pawg ZT2 thiab ZT6. Hauv pawg ZT10 thiab ZT 22, tus naj npawb ntawm cov nas muaj sia nyob yog ob thiab ib qho (40 thiab 20 feem pua), raws li (Fig. 2B). Ua raws li 4-BrC kev tswj hwm, tag nrho cov nas tuag nyob rau hauv 3 d thaum txhaj tshuaj ntawm ZT14 lossis ZT18 (0 feem pua) thiab tsuas yog ib lossis ob (20 lossis 40 feem pua) muaj sia nyob hauv lwm pawg ZT (Fig. 2C). MST yuav luag zoo ib yam nrog cov nas muaj sia nyob hauv txhua pab pawg (Figs. 2D–F). Cov txiaj ntsig no qhia txog kev tuag chronotoxicity ntawm peb BB intermediate metabolites. Tom qab kev tswj hwm ntawm ZT14 (qhov tsaus ntuj), tag nrho peb cov metabolites pom muaj toxicity siab. Interestingly, cov qauv chronotoxicity ntawm cov BB metabolites txawv ntawm cov BB nws tus kheej, uas yog, nas raug rau BB nyob rau hauv lub tsaus ntuj theem muaj rhiab heev rau toxicity.8) Peb yav tas los qhia tias streptomycin, uas qhia nephrotoxicity, yog heev toxicity nram qab no. Qhov tsaus ntuj-theem raug thiab tsis muaj tshuaj lom tom qab lub teeb-theem raug.9) Cov kev tshawb pom ntawm cov kev tshawb fawb yav dhau los no tsis cuam tshuam cov kev tshawb fawb tam sim no, qhia tias qhov sib txawv ntawm cov tshuaj lom ntawm BB thiab peb BB intermediate metabolites raug ntaus nqi rau lawv lub hom phiaj. . BB tsom rau lub siab, thaum nws cov metabolites nruab nrab lub hom phiajlub raum.

Peb tom ntej no tau tshuaj xyuas qhov tshwm sim ntawm lub sijhawm txhaj tshuaj ntawm qhov hnyav ntawm lub cev toxicity siv cov koob tshuaj tsis ua rau tuag ntawm 4-BrC (165 mg / kg). Raws li kev tuag ntxov nyob rau hauv 4 h tau pom nrog 3-BrP thiab BrHQ txhaj, piav raws li saum toj no, peb tsuas xaiv 4-BrC rau cov kev sim tom ntej. Rau kev sim yooj yim, peb xaiv ZT2 thiab ZT14 ua lub sijhawm txhaj tshuaj. Hauv pab pawg ZT2, qib plasma ntawm BUN, qhov taw qhia ntawm nephrotoxicity, tsis tau nce ntxiv tom qab 4-BrC raug; Txawm li cas los xij, qib BUN tau nce ntxiv hauv pawg ZT14 (Fig. 3A). Nyob rau hauv parallel nrog BUN, peb ntsuas plasma qib AST, qhov taw qhia ntawm hepatotoxicity. Peb pom tias 4-BrC kev tswj hwm tsis cuam tshuam rau plasma AST theem ob hauv ZT2 thiab ZT14 (Fig. 3B), qhia tias 4-BrC yog tshuaj lom rau covlub raum. Tsis tas li ntawd, peb kwv yees cov qib ntawm MDA raws li qhov ntsuas oxidative kev ntxhov siab hauv covlub raum. Kev tswj hwm ntawm 4-BrC tseem ceeb nce qib ntawm lub raum MDA hauv ZT14 thiab ZT2 pawg. Txawm li cas los xij, qib MDA hauv pab pawg tshuaj ZT2 tau qis dua li hauv pawg ZT14 (Daim duab 3C).

Txog niaj hnub no, ntau txoj hauv kev tuag ntawm tes xws li apoptosis, necroptosis, thiab ferroptosis tau raug tshaj tawm.11) Tsis tas li ntawd, nws tau raug tshaj tawm tias kev tuag ntawm tes los ntawm 4-BrC feem ntau yog kho los ntawm necrosis.6) Los tshawb xyuas cov hom cell tuag induced los ntawm 4-BrC, peb soj ntsuam cov qib ntawm cov proteins uas muaj feem xyuam rau cell tuag: cleaved caspase-3 rau apoptosis, RIP1/ RIP3 rau necroptosis, thiab GPX4 rau ferroptosis. Tsis muaj kev hloov pauv tau pom tseeb hauv cleaved caspase-3, RIP1, thiab RIP3 qib hauv txhua pab pawg, qhia tias apoptosis thiab necroptosis tsis raug ntxias los ntawm 4-BrC (Fig. 4). Hauv qhov sib piv, qhov txo qis hauv GPX4 qib tau pom tom qab tswj hwm 4-BrC ntawm ZT14 (Fig. 4). Peb tsis tau soj ntsuam qhov 4-BrC-induced raum necrosis los ntawm histopathological tsom xam (cov ntaub ntawv tsis qhia). Qhov no tau qhia tias 4-BrC concentration siv hauv txoj kev tshawb no ua rau lub raum tsis zoo thiab tsis ua rau necrosis. Los ntawm cov txiaj ntsig no, nws tuaj yeem pom tau tias 4-BrC ua rau ferroptosis, uas qhia tau tias muaj kev hloov pauv hauv lub hnub.

Nyob rau hauv txoj kev tshawb no, peb pom BB intermediate metabolites induced chronotoxicity nyob rau hauv lubraum. Hauv qhov sib piv, peb cov txiaj ntsig yav dhau los tau pom BB-induced nephrotoxicity. Txawm li cas los xij, lub raum chronotoxicity tsis tau hloov pauv ntawm lub teeb thiab theem tsaus ntuj.8) Txawm hais tias qhov laj thawj tseem ceeb rau qhov sib txawv ntawm kev tshawb fawb yav dhau los thiab tam sim no tseem tsis tau paub meej, peb xav tias nws tau tshwm sim los npog lub raum chronotoxicity los ntawm kev ua rau mob siab heev. raug mob. Hauv kev sim yav tom ntej, peb yuav tsum ntsuas cov nyiaj tau los ntawm txhua BB-intermediate metabolite vim tias cov nyiaj no tuaj yeem hloov pauv los ntawm CYP thiab / lossis qib raug mob.

Ntau yam kev lom neeg pom tau tias circadian atherosclerosis hauv lawv cov theem qhia. Piv txwv li, Xu et al. tau tshaj tawm diurnal variation nyob rau hauv hepatic antioxidant gene qhia nyob rau hauv nas.12) Glutathione (GSH) yog ib qho antioxidant uas tseem ceeb rau kev tswj kev noj qab haus huv thiab tiv thaiv cov tshuaj lom. Ob leeg 4-BrC thiab 3-BrP tau tshaj tawm kom deplete lub raum GSH qib.4,13) Ferroptosis tau npaj rau xyoo 2012 raws li daim ntawv ntawm cell tuag vim yog elastin, uas inhibits qhov ntshuam ntawm cysteine, ua rau GSH depletion thiab GPX4 inactivation.14) Peb cov txiaj ntsig tau pom tias GPX4 tsuas yog raug cem ntawm ZT14 los ntawm 4-BrC. Yog li, GSH-txog cov proteins lossis cov noob caj noob ces yuav cuam tshuam nrog chronotoxicity.

Los xaus, peb txoj kev tshawb fawb tam sim no qhia tau hais tias BB ua rau lub raum puas thiab puas siab puas ntsws los ntawm nws cov metabolites, xws li 3-BrP, BrHQ, thiab 4-BrC. Yog vim li cas vim li cas lub diurnal variation ntawm nephrotoxicity los ntawm BB tsis tau pom nyob rau hauv peb tsab ntawv tshaj tawm dhau los yog tias lub raum puas tau npog vim muaj zog hepatic puas.

REFERENCES

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Hiroki Yoshioka, a, b, # Sarah Tominaga, a, # Mai Nishikawa, Yasuro Shinohara, Makoto Nakao, Masae Yoshikawa, Tohru Maeda, *, a thiab Nobuhiko Miura*,c

ib lub Tsev Kawm Qib Siab Pharmacy, Kinjo Gakuin University; 2–1723 Omori, Moriyama-Ku, Nagoya 463–8521, Nyiv: b Center for Craniofacial Research, University of Texas Health Science Center ntawm Houston, Tsev Kawm Ntawv Kho Hniav; 1941 East Road, Houston, TX 77054, USA: and cDepartment of Health Science, Yokohoma University of Pharmacy; 601 Momono-Cho, Totsuka-Ku, Yokohama 245–2006, Nyiv. Tau txais Lub Yim Hli 30, 2020; tau txais Lub Kaum Hli 25, 2020

Yog xav paub ntxiv: Ali.ma@wecistanche.com