Determination of Aflatoxin B1–DNA Adduct in Rat Liver by Enzyme Immunoassay
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Determination of Aflatoxin B1DNA Adduct in Rat Liverby Enzyme Immunoassay
T. Vidyasagar, N. Sujatha and R. B. Sashidhar*Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500007, India
A simple, rapid and highly sensitive indirect competitiveenzyme-linked immunosorbent assay (ELISA) todetermine aflatoxin B1 (AFB1)DNA adducts is reported.Polyclonal antibodies specific to the aflatoxinB1N7guanine adduct were produced using a novelsynthetic antigen, bovine serum albumin (BSA)guanineAFB1. The antibodies were characterized by theOuchterlony double diffusion technique and by antibodycapture assay. The working range of the indirectcompetitive assay developed was between 0.45 and 330 ngof the analyte [calf thymus (CT)DNAAFB1]. A 50%inhibition was attained at 15 ng of the analyte(CTDNAAFB1). The antibody capture assay indicatedthat the antibody produced cross-reacted 100, 92 and110% with BSAguanineAFB1, CT-DNAAFB1 andCT-DNAformamidopyrimidineAFB1, respectively.When free AFB1 and guanine were used as competinganalytes, the antibodies showed @5% and zerocross-reactivity at the 50% inhibition level. Spikingstudies indicated a recovery in the range 9697 and7478% when standard CT-DNAAFB1 was added to10 mm phosphate buffer (pH 7.2) and control rat livertissue, respectively. Rats exposed to a single oral dose of1 mg kg21 body mass of pure AFB1 were used to validatethe method. The AFB1DNA adduct formed in the livertissue after 48 h of exposure was determined using theELISA method developed. The liver AFB1DNA adductranged between 6.06 and 7.94 mg mg21 DNA. Theproposed method may find application in the biologicalmonitoring of aflatoxin B1 in molecular epidemiologicalstudies to assess the dietary exposure of aflatoxins.Keywords: Aflatoxin B1; calf thymus DNAaflatoxin B1;aflatoxin B1N7guanine; calf thymusDNAformamidopyrimidineaflatoxin B1; aflatoxin B1DNA;enzyme-linked immunosorbent assay
Aflatoxins are potent hepatotoxic and hepatocarcinogeniccompounds produced by Aspergillus flavus and Aspergillusparasiticus species. A variety of human foods such as cereals,millets and oil seeds are susceptible to these ubiquitous fungi,which infect and produce aflatoxins during growth, harvest,transport and storage.1,2 Aflatoxin B1 (AFB1) is one of the mostpotent carcinogens and is classified as a Group I carcinogen bythe International Agency for Research on Cancer (IARC, Lyon,France).2 Exposure to AFB1 has been associated with anincreased incidence of primary hepatocellular carcinoma(PHCC), which is the seventh most frequent cancer in the worldand particularly in South-East Asia, China and Sub-SaharanAfrica.37
After gaining entry into the systemic system through the diet,AFB1 is metabolically activated by liver microsomal enzymes(cytochrome P450-dependent enzymes) into a highly reactiveelectrophilic species, an 8,9-epoxide, which efficiently binds tonucleophilic sites of cellular macromolecules.810 The carcino-genic 8,9-epoxide specifically binds at the N7-position of
guanine in DNA or attacks the e-amino group of lysine inproteins.8,9,11,12
In the recent past, biological monitoring of the AFB1N7-guanine adduct has been used as a molecular dosimeter ofexposure to AFB1 in molecular epidemiological studies.1316Various analytical methods have been developed for thedetection and determination of the guanineAFB1 adduct fromhuman and animal tissues, including chromatographic, im-munological and immunocytochemical methods.1518 How-ever, all these methods were based on antibodies which wereproduced to the AFB1, moiety. An AFB1-specific monoclonalantibody affinity chromatographic method coupled with high-performance liquid chromatography(HPLC) was successfullyused to detect AFB1N7-guanine from acid-hydrolysed DNAsamples in human tissues after acute poisoning with AFB1.17Similarly, a multiple monoclonal antibody, specific to aflatoxinB1, has been used in affinity chromatography along with HPLCfor the detection of AFB1N7-guanine in rat urine.16 Animmunoanalytical method was also developed in which mono-clonal antibodies raised against AFB1formamidopyrimidinewas used to detect AFB1formamidopyrimidine adducts inDNA samples from rat liver tissues.15 A sensitive im-munocytochemical method for the quantification of AFB1bound to DNA in various rat tissues using AFB1-specificmonoclonal antibodies has also been reported.18
This paper reports an in vitro method to determine AFB1N7-guanine in intact DNA by using AFB1N7-guanine-specificpolyclonal antibodies. An indirect competitive ELISA wasdeveloped to determine the AFB1N7-guanine adduct in DNAextracted from liver tissues of rats exposed to a single oral doseof AFB1.
A DU-50 recording spectrophotometer (Beckman, Fullerton,CA, USA) was used for spectral analysis. An SLT-Spectra IImicroplate reader (Grodig, Salzburg, Austria) was used tomeasure the optical density. Polystyrene microtitre ELISAplates were purchased from Greiner (Nurtingen, Germany).
AFB1, bovine serum albumin (BSA) (fatty acid free, RIAgrade), dimethyl suberimidate, guanine, calf thymus (CT)DNA, Freunds complete adjuvant (FCA), Freunds incompleteadjuvant (FIA), anti-rabbit immunoglobulin G (IgG) labelledwith alkaline phosphatase raised in goat (whole molecule),gelatin, trinitrobenzenesulfonic acid (TNBS) and polyestersilica gel G TLC plates (20 cm 3 20 cm; particle size 225 mm)were purchased from Sigma (St. Louis, MO, USA). m-Chloroperbenzoic acid (MCPBA) was purchased from Merck(Darmstadt, Germany). All other chemicals were of analytical-reagent grade.
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Two male rabbits of New Zealand white strain (16 weeks old;2.53.0 kg) and Fischer 344 strain rats (body mass 150220 g)were procured from the National Institute of Nutrition (Hyder-abad, India)
Aflatroxin B1 and many of its derivatives are potentiallycarcinogenic and great care should be exercised to avoidpersonnel exposure. When handling the pure compound in thecrystalline form, the use of disposable cotton gloves isrecommended. The general care for minimizing exposure tochemical carcinogens and for reducing the risk of laboratorycontamination was followed as recommended by Montesanoet al.19 and according to National Institutes of Health (NIH)guidelines.20
Synthesis of the Antigen BSAguanineAFB1
The antigen BSAguanineAFB1 was synthesized in two steps.First, the BSAguanine conjugate was synthesized using thehomobifunctional reagent dimethyl suberimidate (DMS), re-sulting in the covalent linkage of e-amino groups of BSA withthe 2-amino group of guanine.21 The BSAguanine conjugatewas then reacted with AFB1-8,9-epoxide generated by reactionof MCPBA with pure AFB1, resulting in the adduction of AFB1at the N7-position of guanine.22 The molar ratio of BSA toguanineAFB1 was determined by TNBS assay.23 The synthe-sized BSAguanineAFB1 conjugate was characterized byTLC. Polyester silica gel TLC plates were used and an aliquotof the BSAguanineAFB1 in 100 mm phosphate buffer (pH7.2) was spotted along with standard AFB1 in chloroform. Theplate was developed in chloroformacetone (9 + 1) as themobile phase and was viewed in a longwave UV (365 nm)chamber.
Synthesis of Calf Thymus DNAAFB1 adduct
The CT-DNAAFB1 adduct was synthesized as described byIyer et al.11 using 2 mg of CT-DNA and 400 mg of AFB1 withMCPBA as the oxidizing agent in a biphasic reaction [dichloro-methane100 mm phosphate buffer, (pH 7.2) (1 + 1, v/v)]. Thisadduct was used as a standard reference material and also as acoating antigen on microtitre plates. The molar ratio of CT-DNA to AFB1 was determined by spectral analysis of theunreacted AFB1 in dichloromethane and CT-DNAAFB1 in thebuffer fraction at 360 nm.
Production of Polyclonal Antibodies Against the AntigenBSAguanineAFB1
Two male rabbits (2.53.0 kg) were chosen for the productionof polyclonal antiserum against BSAguanineAFB1. A primerdose of BSAguanineAFB1 equivalent to 60 mg of AFB1 perkg body mass of the animal was given using FCA by themultiple site epidermal injection technique.24 The subsequentboosters were given in FIA by the intra-muscular route after 4weeks. The dose of the antigen for the first booster wasequivalent to 45 mg of AFB1 per kg body mass and the secondto 30 mg of AFB1 per kg body mass for the next two boosters.Each booster was spaced by 1014 days. Test bleeding wascarried out using a heparinized micro-capillary from the retroorbital plexus. At the end of the immunization schedule theanimals were killed and the blood was collected by cardiacpuncture. Serum was separated out, lyophilized and stored at220 C until further use.
Characterization of Antibodies
The serum from each animal was analysed for the presence ofantibodies against the antigen BSAguanineAFB1 by theOuchterlony double diffusion technique.25
Titre Determination of Antisera by Antibody Capture Assay
Microtitre plate wells were coated with 500, 750 and 1000 ng ofCT-DNAAFB1, which were equivalent to 10.66, 16 and 21.32ng of AFB1, respectively, in 50 ml of 100 mm phosphate buffer(pH 7.2). The plate was dried overnight at 37 C before washing(33) with 10 mm phosphate-buffered saline containing 0.05%Tween-20 and 0.01% sodium azide (PBS-T). The wells wereblocked for non-specific binding by incubation with 50 ml of0.1% gelatin in 10 mm PBS (pH 7.2) for 30 min at 37 C. Afterwashing (33) the wells with PBS-T, diluted BSAguanineAFB1 antiserum (1 3 1032 3 105 dilution) was added (50 mlper well; antiserum was diluted with 10 mm PBS (pH 7.2)containing 0.01% BSA). The plate was incubated at 37 C for 2h and subsequently washed (33) with PBS-T. The wells werethen dispensed with 50 ml of a 1 : 5000 dilution (in 10 mmphosphate buffer, pH 7.2) of alkaline phosphatase-labelled anti-rabbit IgG raised in goat (second antibody). The plate wasincubated for 1 h at 37 C before washing (33) with PBS-T.Substrate solution containing 1.25 mg ml21 of p-nitrophenylphosphate and 0.05 mm MgCl2 in 10% diethanolamineHClbuffer (pH 9.6) was then added (150 ml per well). The reactionwas terminated after a 45 min incubation at 37 C by adding100 ml of 5 m NaOH to each well. The absorbance wasdetermined at 405 nm using a microplate reader against CT-DNA blank. The optimum antibody titre was determined bychecker board analysis in which different concentrations of CT-DNAAFB1 (500, 750 and 1000 ng per well) were coatedvertically and different dilutions of the antiserum raised againstBSAguanineAFB1 were dispensed into each well horizon-tally in a 96-well (8 rows 3 12 columns) microtitre plate.
Validation of the Method
Fischer 344 rats, four male (body mass 180220 g) and fourfemale (body mass 150170 g) were given 1 mg kg21 bodymass of pure AFB1 dissolved in peanut oil by a single oral dosethrough gavage. A control group of four male and four femalerats were given only the peanut oil vehicle. All the rats wereprovided with a commercial powder diet containing 20%protein, 65% starch, 5% fat, vitamins and minerals. Diet anddistilled water were given ad libitum. The rats were killed bycervical dislocation 48 h after treatment. The livers wereexcised, rinsed with ice cold saline and weighed. Liver DNAwas extracted by the method of Groopman et al.26 The purity ofthe extracted DNA was determined by the 260/280 nmabsorbance ratio, followed by the determination of DNA by thediphenylamine method.27
Spiking studies were also carried out in liver samplesobtained from control rats and in 10 mm phosphate buffer (pH7.2) using standard CT-DNAAFB1. Spiking was done at twodifferent levels of 20 and 50 mg g21 liver and 0.2 and 1.6 mgml21 buffer. The liver DNA was extracted and the amount ofCT-DNAAFB1 was determined by indirect competitiveELISA. The repeatability (within-assay variation) and thereproducibility (between-assay variation) of the immunoassaywere also validated.
Indirect Competitive ELISA for Quantification ofAFB1DNA adduct
The indirect competitive ELISA is based on the principle ofcompetition between the immobilized ligand and the free ligand
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for the limited binding sites present on the ligand-specificantibodies (primary antibodies) in the assay system. Theamount of the primary antibodies bound to the immobilizedligand is detected by an enzyme-labelled anti-species antibodies(second antibodies). Thus, the net enzyme activity associatedwith the second antibody is indirectly proportional to thevarying concentration of the free ligand (i.e., the higher theconcentration of the free ligand, the lower is the enzymeactivity).
Microtitre plate wells were coated with 750 ng equivalent ofCT-DNAAFB1 in 100 mm phosphate buffer (pH 7.2). Thewells were blocked with 0.1% gelatin in 10 mm PBS (pH 7.2)for 30 min at 37 C to prevent non-specific binding. The platewas washed (33) with PBS-T and different concentrations ofstandard CT-DNAAFB1 in 25 ml of distilled water (pHadjusted to 7.2) ranging from 0.45 to 330 ng along with knownconcentrations of DNA extracted (23 mg equivalent) fromdifferent rats in 25 ml of distilled water (pH adjusted to 7.2) wereadded to different wells. Titre determination of antiserum byantibody capture assay showed that a 1 : 7000 dilution was theoptimum antibody titre for a coating antigen concentration of750 ng per well. Hence diluted antiserum (1 : 3500) raisedagainst BSAguanineAFB1 in 20 mm PBS (pH 7.2) containing0.02% BSA was added to each well (25 ml per well) uniformlyto give a final antiserum dilution of 1 : 7000. The plate wasincubated at 37 C for 2 h before washing (33) with PBS-T. Tothe washed wells, 50 ml of 1 : 5000 diluted second antibody(alkaline phosphatase-labelled anti-rabbit IgG) in 10 mm PBS(pH 7.2) were added. The rest of the protocol was similar to thatfor titre determination as detailed above. The absorbance wasdetermined at 405 nm using a microplate reader (along with areagent blank). The percentage binding of antibodies versusconcentration of the analyte (CT-DNAAFB1) was used togenerate an inhibition plot, based on linear regression analy-sis.
Cross-reactivity studies of the Antiserum with the AntigenCT-DNAformamidopyrimidineAFB1
CT-DNAAFB1 adduct was converted into CT-DNAfor-mamidopyrimidineAFB1 by incubation with 100 mm carbo-nate buffer (pH 9.6) for 2 h at 37 C as described by Groopmanet al.28 Base treatment of CT-DNAAFB1 results in ringopening of the imidazole of the guanine residues and hence theformation of CT-DNAformamidopyrimidineAFB1. The CT-DNAformamidopyrimidineAFB1 thus generated was used asa coating antigen in the ELISA to check the cross-reacti...