Arch Toxicol (2006) 80: 572–579 DOI 10.1007/s00204-006-0076-2
O RG AN T OX IC ITY A N D M E CH AN I SM S
Faezeh Fatemi Æ Abdolamir Allameh Æ Abolfazl Dadkhah Mehdi Forouzandeh Æ Somayeh Kazemnejad Roya Sharifi
Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1 Received: 28 September 2005 / Accepted: 23 January 2006 / Published online: 24 February 2006 Springer-Verlag 2006
Abstract The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GSTP activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50–60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/ kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in
F. Fatemi Æ A. Allameh (&) Æ A. Dadkhah Æ S. Kazemnejad R. Sharifi Department of Biochemistry Faculty of Medical Science, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran E-mail:
[email protected] Tel.: +98-21-88011001 Fax: +98-21-88006544 M. Forouzandeh Department of Biotechnology Faculty of Medical Science, Tarbiat Modarres University, P.O. Box 14115-331, Tehran, Iran
fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to transplancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation. Keywords Aflatoxin B1 Æ Glutathione transferase Æ Prenatal Æ Postnatal Æ mRNA expression
Introduction Aflatoxin B1 (AFB1) is a mycotoxin produced as a secondary metabolite by the toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. These fungi are ubiquitous and can grow on a wide variety of food commodities (Wilson and Payne 1993). Hepatotoxicity and hepatocarcinogenicity of AFB1 are well recognized in different animal species (Herrold 1969; Newberne and Butler 1969; Moore et al. 1982). The maternal dietary AFB1 exposures during pregnancy may lead to transplancental AFB1 teratogenicity and carcinogenicity in animal models (Grice et al. 1973; Goertller et al. 1980; Elis and DiPaolo 1967). Furthermore, evidences show that transplancental transfer of AFB1 in humans may increase the risk of childhood cancer (Groopman et al. 1986; Denning et al. 1990; Wild et al. 1992; Autrup 1993). AFB1 exerts its biological effects after metabolic activation by cytochrome P-450-dependent monooxygenase to its reactive form i.e. AFB1-8,9-epoxide (AFBO) which then interacts with cellular macromolecules, particularly DNA (Swenson et al. 1977; Essigman et al. 1982). Several studies have shown that AFB1epoxide could be inactivated for carcinogenesis by cytosolic glutathione (GSH) S-transferases (GSTs) forming AFB1-GSH conjugate (Coles et al. 1985; Neal et al. 1987; Jhee et al. 1988). In vitro and in vivo studies show that GSTs play a crucial role in modulation of
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AFB1–DNA adducts formation (Raj et al. 1986; Ramsdell and Eaton 1990). AFBO–GSH conjugate formation catalyzed by GST is the major route of AFB detoxification and a major factor in determining species resistance (Ramsdell and Eaton 1990; Neal et al. 1981). GSTs (EC: 2. 5. 1. 18) are a family of enzymes involved in detoxification of foreign compounds. GSTs exist as homo-or hetero-dimers and based on the sequence similarities, mammalian cytosolic GSTs have been grouped into at least seven classes viz. alpha, mu, pi, theta, sigma, omega and zeta (Strange et al. 2000; Hayes et al. 2005). Developmentally regulated expression of GSTs is of particular interest and the ability of the fetal liver to detoxify transplancental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicology. According to Hayes et al. (1994), fetal liver possesses substantial levels of GST activity toward AFBO. The enzyme responsible for this activity is the alpha-class GST heterodimer comparising Yc and Yc2 subunits, this subunit is now called GSTA5 (Hayes et al., 2005). Placental GST i.e., GST-P is the main isoform in normal placental tissue and comprises 36% of total GST activity (Steisslinger and Pfleiderer 1988) and 67% of the total GST concentration (Zuslerzeel et al. 1999). GST-P expression is relatively higher in fetal liver as compared to that in normal adults and its concentration is slowly decreases after birth. GST-P is expressed in fetal liver and gradually decreases in concentration during development and normally absent in adult liver. Earlier we demonstrated that there is difference in the rate of AFB1-DNA adduct formation in liver tissues of growing and adult rats treated with AFB1 (Chelcheleh and Allameh 1995). Accordingly the rate of GSTdependent AFB–GSH conjugate formation in liver and kidneys of weanling and adult rats was found to be different (Allameh et al. 2000). Influence of AFB1 on the expression of GST-P at protein and mRNA levels during fetal and postnatal stages may be implicated in susceptibility/resistance to AFB1 carcinogenicity or teratogenicity. Hence, in this study experiments were carried out to investigate the effects of AFB1 on expression of rat liver GST with emphasis on GST-P at protein and mRNA levels in liver tissues before and after birth.
Hyperfilm-ECL and Hybond-C extra nitrocellulose membrane were the products of Amersham Life Science, UK. Primers for b-actin and GST-P genes were prepared by Farayand-Danesh Co., Tehran, Iran. All other chemicals and reagents were of analytical grade locally available.
Animals and treatments Prenatal experiments Albino rats of Wistar strain were used throughout this study. Animals were obtained from Pasteur Institute of Iran. They were allowed to acclimatize to their surrounding for 1 week. They were fed standard pellets and water ad libitum. After acclimatization period, male and female rats were housed separately in cages for mating. Females were checked for pregnancy every morning by conventional vaginal smear test. In each set of experiment, pregnant rats (n=20) were selected and on day 8.5 of gestation, one group (n=4) received single i.p. dose of AFB1 (0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) and a matching control group (n=4) received equal volume of the vehicle alone i.e. DMSO. On day 19 of gestation, 1 day before delivery, the mothers undergone cesarean operation under slight diethyl ether anesthesia. The uterus was removed, transferred to a Petri dish, and combined with phosphate buffer, 100 mM, and pH 7.0. Implantations were counted; each gestation sac was opened and the content examined under dissecting scope. The teratogenic effects of AFB1 on the number of embryos were recorded. The number of normal living fetuses with no gross malformation, malformed embryos with gross malformation and dead or reabsorbed fetuses was recorded. Livers were removed carefully and homogenized in phosphate buffer, 100 mM, and pH 7.0 to obtain a 20% homogenate (w/v). Cytosolic fraction of liver was prepared by ultracentrifugation as described previously (Allameh et al. 1987). Liver cytosolic fractions were divided into aliquots and stored at 70C for further use. Postnatal experiments
Materials and methods Chemicals:Aflatoxin B1 (AFB1), acrylamid and bisacrylamid, anti-glutathione S-transferase-P (anti-GST-P), CDNB, bovine serum albumin (BSA), GST-P, dimethyl sulfoxide (DMSO), ethacrynic acid (ETA), anti-IgG conjugated to horse radish peroxidase (anti-IgG-HRP), RNase, diethyl pyrocarbonate (DEPC) were from Sigma chemical Co., St. Louis, USA. RNasin, Reverse transcriptase (moloney murine leukemia virus (M-MuLV), oligo-dt mixture and Taq DNA polymerase were purchased from Sina-gene, Tehran, Iran. ECL-plus reagent,
In this experiment, growing rats (14±2 days old) weighing 24–28 g were used. Each set of experiment comprised of two groups. Each rat in the treated group was injected a single i.p. dose of AFB1 (2.0 or 3.0 mg/kg bw) prepared in DMSO. Rats in control group received vehicle alone i.e. DMSO. The treated and its matching control groups were sacrificed at different time intervals (2, 6, 12, 24, 36, and 48 h after injection). Livers were removed, washed in ice-cold phosphate buffer (100 mM, pH 7.0), and homogenized in the same buffer to obtain a 20% w/v tissue homogenate. Cytosolic fraction was prepared by
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ultracentrifugation, divided into aliquots and stored at 70C for further use. Estimation of cytosolic GST activity: Liver cytosolic GST was measured spectrophotometrically according to the procedures described by Habig et al. (1974). GST activity was measured using CDNB (a general substrate) and ETA (specific substrate towards GST-P).
The mixture was mixed and incubated for 5 min at 70C. In order to prepare cDNA from total RNA, 4 ll of PCR buffer (5·), 0.5 ll of MgCl2 and 0.5 ll RNasin and 1 ll of AMV reverse transcriptase were added to the reaction mixture, incubated at 42C for 60 min. PCR reaction: Specific primers for GST-P and b-actin were used. The characteristics of the primers used are as follows:
Western blot analysis of GST-P
Rat GST-P Forward: 5¢-CCT CAC CCT TTA CCA ATC TA-3¢. (NCBI Accession number Xo.2904).
Western blotting technique was performed based on the procedure of Towbin et al. (1979). For this purpose, cytosolic proteins were resolved on 12% acrylamide gel (SDS-PAGE). Proteins were electrophoretically separated at a constact voltage of 100 V in 25 mM Tris–HCl pH 8.3 containing SDS and glycine. Proteins were blotted onto nitrocellulose membrane (45 lm pore size) at 100 V for 1 h on a Protean blotter system (Bio-Rad) using 25 mM Tris, pH 8.3, 192 mM glycine and 15% v/v methanol. After transfer, the membranes were rinsed in Tris-buffered saline with Tween (TBS-T), blocked in 10% skimmed milk/TBS-T, rinsed three times with TBST and incubated for 60 min in primary antibody (antiGST-P). Following three rinses with TBS-T, membranes were incubated for 60 min in horseradish peroxidaseconjugated secondary antibody. The blots were rinsed with TBS-T and visualized using the Amersham ECLPlus chemiluminescence system, and quantitation of the western blots was accomplished using densitometric scanning (LabImage Software, version 2.6; Kapelan, GmbH Co., Germany) of the results of autoradiographs. Relative density of immunoreactivity was measured using Adobe Photoshop, version 7.0 for Windows (Adobe Systems Incorporation, San Jose, CA, USA).
Isolation of RNA and cDNA synthesis Total RNA was isolated using phenol extraction mixture e.g., RNX (Sinagene, Tehran, Iran). Briefly, 50–100 mg fresh liver was homogenized in 1 ml of the extraction mixture and left at room temperature for 5 min. Then 200 ll of chloroform added and gently mixed. The mixture was centrifuged at 12,000g for 15 min at 4C. The supernatant (1 ml) containing nucleic acids was separated and transferred to a fresh micro tube. To this, an equal volume of iso-propanol (prepared in DEPCtreated water) was added and centrifuged for 10 min at 12,000g. RNA was suspended in 1 ml ethanol (75%) and centrifuged again at 7,500g for 5 min at 4C. Finally RNA was dissolved in 50 ll DEPC-treated water and checked spectrophotometrically (OD=260 nm) and loaded on agarose gel electrophoresis. The first strand cDNA was synthesized from the isolated RNA using oligo-dt primers. The reaction mixture contained; 5 lg RNA; 5 ll (10 pmol) oligo-dt; 3 ll deionized water and 1 ll (10 mM) dNTP mixture.
Rat GST-P Reverse 5¢-TTC GTC CAC TAC TGT TTA CC-3¢. Molecular weight=5,994 g/mol, Tm=58C, GC percentage=45. The region was amplified with this pair of the primers was from nucleotides 129 to 680 of the rat cDNA. For b-actin Forward: 5¢-TAC GTA GCC ATC CAG GCT GTG-3¢. Reverse: 5¢-GAT CTT GAT CTT CAT GGT GCT AGG-3¢. These primers amplify a 590 bp fragment corresponding to bases 400 to 990 of b-actin. (NCBI Accession number NM-031144). PCR amplification of GST-P and b-actin Five microliter of the cDNA (reverse transcription mixture) and oligonucleotides at a final concentration of 20 lM of primers were analyzed in a 50-ll volume. PCR amplification for the target sequence (GST-P) and internal control (b-actin) for each sample were performed. First strand cDNA derived from the tissue was directly used to amplify the target sequence (GST-P) and internal control (GST-P) genes for each sample. For this, 5 ll of the first strand cDNA at a final concentration of 20 lM of primers were used in a 50 ll total volume. The reaction mixture was subjected to the following amplification protocol; 1 cycle at 90C for 150 s for initial denaturation and 1 cycle at 95C for 10 min., followed by 35 cycles at 95C for 30 s (denaturation) and 55C for annealing and at 72C for 1 min for extension. The reaction continued for 10 min at 72C for final extension. Other assays: Protein concentration of plasma samples was estimated by the method of Bradford (1976) using BSA as standard. Statistical analysis All the samples and standards were run in duplicate; the results are presented as mean ± SEM. Differences between control, and treated animals were analyzed using Student’s t test, considering P
