Cancer Letters, 56 (1991)
Elsevier Scientific Publishers Ireland Ltd.
Enzyme immunoassay for (3-amino-l-methyl-5H-pyrido[4,3-blindole), pyrolysate T.Nambara
S. Miyairi and Pharmaceutical
(Received 20 September 1990) (Revision received 31 October 1990) (Accepted 26 December 1990)
of the cancer risk by the
pyrolysates a sensitiue enzyme imfor S-amino-l -methyl-5H-pyrido-
munoassay [4,3-blindole (Trp-P-Z), a mutagenic and carcinogenic substance, found in foods and biologicalfluids has been developed. The dose-
response curue obtained was suitable for the determination
in the range of ZO-
pg. The immunoassay ed to be satisfactorily reactioity
specific as judged by cross-
Trp-P-2 levels in the basic and neutral fraction derived from pyrolyzed amino acids and related compounds.
Keywords: [4,3-blindole; immunoassay
Correspondence Chemistry, Aobayama,
3-amino-l-methyl-5H-pyrido tryptophan pyrolysate; enzyme
to: S. Miyairi, Ph.D.,
Pharmaceutical Sendai 980.
Published and Printed in Ireland
of Analytical University,
Since the mutagenicity of Salmonella typhimurium was found in cooked foods [ 121, a number of mutagenic compounds have been isolated from the char of broiled meat and fish, and pyrolytic tar of amino acids . Both 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole and 3-amino- 1-methyl-5H(Trp-P- 1) pyrido[4,3-blindole (Trp-P-2) derived from pyrolyzed tryptophan were characterized as potent mutagens . Furthermore, carcinogenicity was also found in both compounds [4,9]. Determination of the pyrolysates in foods is required for evaluating cancer risk epidemiologically. For this purpose several methods have been chromatographic developed. However, clean-up procedures for the pyrolysates formed from foods are somewhat troublesome in gas or liquid chromatographymass spectrometry [ 1,161 and high-performance liquid chromatography [8,15]. On the other hand, immunoassay is convenient and useful for the epidemiological study. This method appears to be suitable for the routine assay of a specific marker component in the complicated matrix without tedious pretreatments. The present paper deals with development of
Elsevier Scientific Publishers Ireland Ltd
enzyme immunoassay plicabilities.
and its ap-
C,,H,,N,O,: C, 65.58; H, 5.50; N, 13.50. Found: C, 65.61; H, 5.52; N, 13.24. NMR 2.78 (CDCl,/CD,OD) 6: (4H, s,
Materials and Methods COCH,CH,CO), Materials Trp-P- 1, Trp-P-2 and other y-carboline derivatives [ 141, 3-amino-5H-pyrido[4,3-b]indole, 3-amino-l-ethyl-5H-pyrido[4,3-blindole and 3-amino-4-methy1-5H-pyrido[4,3-b]indole, were generous gifts from Dr. M. Okada of Tokyo Biochemical Research Institute and Prof. K. Shudo of University of Tokyo. 3-Maleimidobenzoyl chloride was prepared by the method of Monji et al. [ 111. ,&Galactosidase (EC 184.108.40.206) from Escherichia co/i (360 units/mg protein) and bovine serum albumin (BSA) were supplied by Sigma Chemical Co. (St. Louis, MO, U.S.A.). o-Nitrophenyl-P-D-galactopyranoside, goat anti-rabbit IgG antiserum and normal rabbit serum were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and Daiichi Radioisotope Labs. Ltd. (Tokyo, Japan), respectively. IH-NMR spectra were recorded on a JEOL FX-100 spectrometer at 100 MHz using tetramethylsilane as an internal standard. Abbreviations used are s = singlet and m = multiplet. Melting points were measured on a Yanagimoto hot stage apparatus and are uncorrected. Trp-P-2 N- (3-carboxypropionylamide) (3) A solution of Trp-P-2 (1) (100 mg) and succinic anhydride (100 mg) in pyridine (3 ml) was heated at 65-75OC for 2 h. After the excess reagent was decomposed by the addition of water (1 ml), the resultant mixture was acidified with 0.1 N HCI and extracted with n-butanol. The organic phase was washed with watersaturated n-butanol and then evaporated down. The residue dissolved in MeOH was treated with diazomethane-etherate in the usual manner. The crude product was purified by a column silica chromatography on with gel CH2C12-MeOH. Recrystallization of the eluate from MeOH gave the Whemisuccinate methyl ester (2) (68 mg) as pale yellow leaflets. Melting point: 206-207°C (decomp.). Anal. calcd. for
2 N II
3.73 (3H, s, OCH,), 7.14-7.56 and 7.92-8.10 (3H and 2H, m, aromatic). To a solution of 2 (65 mg) in 95% EtOH (10 ml) was added 10% NaOH (0.4 ml), and the mixture was stirred at room temperature for 1 h. After neutralization with 2 N HCI, the resulting solution was concentrated to approximately 1 ml under reduced pressure. A pale yellow precipitate (60 mg) was collected by filtration, washed with water and dried. Compound 3 gradually changed to brown in color above 250°C, but did not melt even at 3OOOC. NMR (DMSO-d,) 6: 2.50-2.69 (4H, m, COCH,CH,CO),
2 'N II
(3H and 2H, m,
Trp-P-2 N-(3-Maleimidobenzoylamide) (4) A mixture of 1 (65 mg), 3-maleimidobenzoyl chloride (400 mg) and anhydrous AcONa (500 mg) in EtOAc (20 ml) was refluxed for 8 h. The precipitate formed was removed off by filtration through silica gel on a sintered glass funnel and washed with CHCl,-MeOH (5: 1). The filtrate and washings were combined and evaporated down. The residue was rinsed with acetone to remove 3-maleimidobenzoic acid. Reprecipitation of the residue from aqueous EtOH gave 4 (100 mg) as pale yellow amorphous powder. Melting point 267-27OOC. Anal. calcd. for C2,H,,N,03. HCl l/2 H20: C, 62.51; H, 4.11; N, 12.68. Found: C, 62.51; H, 3.78; N, 12.44. NMR (DMSO-d,) 6: 3.13 (3H, l
(6H and 5H, m,
ConjugationofTrp-P-2 N-(3-carboxypropionylamide) with BSA A solution of 3 (25 mg), l-ethyl-3-(3dimethylamino-propyl)carbodiimide HCI (30 mg) and N-hydroxysuccinimide (18 mg) in 95% DMSO (0.5 ml) was stirred at room temperature for 8 h. The resultant solution was diluted with EtOAc, washed with water, dried over anhydrous Na$O,, and evaporated down. The structure of the residue was characterized as the activated ester by NMR spectrum. NMR (DMSO-d,) 6: 2.84 (7H, s, COCH,CH,CO l
7.24-7.70 and 8.12-8.26 (3H and 2H, m, aromatic). A mixture of the activated ester (25 mg) and BSA (75 mg) in 0.05 M phosphate buffer (pH 7.3)/pyridine (l:l,v/v) (1.2 ml) was stirred at 4OC for 10 days. The BSA fraction was precipitated by addition of acetone followed by centrifugation at 3000 rev. /min for 10 min. This procedure was repeated until unconjugated TrpP-2 derivative was removed completely. The precipitate was dissolved in 80% aqueous pyridine and dialyzed against cold running water overnight. Lyophilization of the resultant solution gave the Trp-P-2-BSA conjugate (approx. 50 mg) as fluffy powder. The number of Trp-P-2 molecules incorporated into a BSA molecule was elucidated to be 10 by UV spectrophotometric analysis. Preparation of anti-Trp-P-2 antiserum The Trp-P-2-BSA conjugate (1 mg) dissolved in sterile isotonic saline (0.5 ml) was emulsified with complete Freund’s adjuvant (0.5 ml). The emulsion was injected into a domestic male albino rabbit subcutaneously at multiple sites along the back. This procedure was repeated once every fortnight. Blood was
withdrawn at 6 months after the first administration and centrifuged at 3000 rev./min for 10 min. The serum separated was stored at 4OC with 0.1% sodium azide. Preparation of Trp-P-2 N-(3-maleimidobenzoylamide)-P-galactosidase conjugate Compound 4 (4.5 or 9.0 pg) in DMSO (10 ~1) was added to 0.05 M phosphate buffer (pH 6.0) (0.5 ml) containing /3-galactosidase (500 pg). The reaction mixture was immediately vortex-mixed, then allowed to stand overnight at 4OC with occasional shaking. The resultant solution was dialyzed against cold 0.05 M phosphate buffer (pH 7.3) (3 x 11) for 2 days, diluted with buffer to the concentration of 500 pg/ml with 0.5% BSA, and stored at 4OC. The Trp-P-2 enzyme conjugate prepared by this method was stable for several months as regards enzymic activity and immunoreactivity under this storage condition. For the immunoassay, this stock solution was diluted with 0.05 M phosphate buffer (pH 7.3) containing 0.9% NaCl and 0.1% gelatin (buffer A) containing 0.5% normal rabbit serum. Procedure for enzyme immunoassay Trp-P-2-enzyme conjugate (0.1 pg, 0.1 ml) was mixed with Trp-P-2 standard (0, 20, 50, 100, 200, 400, 800 pg) or the test sample in buffer A (0.1 ml) and diluted antiserum (0.1 ml) sequentially, and was allowed to stand at 4OC for 4 h. Then, to the mixture was added goat anti-rabbit IgG antiserum (0.1 ml) diluted to 1:30 with the buffer A containing 0.3% ethylenediaminetetraacetic acid, and allowed to stand at 4OC for 16 h. The resultant mixture was diluted with Buffer A (1.5 ml) and centrifuged at 3000 rev./min for 10 min. The immune precipitate collected by aspirating off the supernatant was washed with the buffer A (1.5 ml) once by the same procedure and then used for measurement of the enzymic activity. Measurement of /3-galactosidase actiuiry The immune precipitate was suspended into the Buffer A (1 ml) containing 0.2% magnesium chloride and 0.7% 2-mercaptoethanol, in-
cubated at 37OC for 3 min and then added onitrophenyl-O-D-galactopyranoside (0.06%) 1 ml) in 0.05 M phosphate buffer (pH 7.3). The mixture was incubated at 37OC for 90 min, and added 1 M sodium carbonate (2 ml) to terminate the reaction. The absorbance at 420 nm was measured. Determination immunoassay
of the enzyme
The specificity of the assay system was assessed by cross-reactivity to four y-carboline derivatives and two amino acids related to TrpP-2. The cross-reactivity was expressed as percentage of the amount of Trp-P-2 which reduced the enzyme activity in the precipitate by half to that of the compound listed in Table I. The specificity was also assessed using extracts of various amino acid pyrolysates prepared by the follwing procedure. Amino acid or its related compound (100 mg) was pyrolyzed by a direct gas flame. The volatile product was trapped into ice-cooled MeOH. The resultant tar and trapped volatile substances were combined, filtered and evaporated down. The residue dissolved in 1 N HCl (2 ml) was adjusted pH above 10 by 6 N NaOH, and extracted twice with ethyl ether (5 ml). The pooled ether-soluble fraction was evaporated down and the MeOH (2.5 ml) solution of the residue (the basic and neutral fraction) was subjected to the enzyme immunoassay. Results and Discussion The primary amino group present in Trp-P-2 was utilized for haptenic derivatization (Fig. 1). The compound 3 produced from Trp-P-2 by the reaction with succinic anhydride was purified via the methyl ester. The hapten (3)BSA conjugate was prepared as the immunogen by the activated ester method using Nhydroxysuccinimide. The appropriate antiserum was obtained from rabbits at 6 months after the first administration of the immunogen. Contrary to the expectation, the reactivity of 3 was insufficient in the mild condition for providing the enzyme conjugate used as the labeled antigen
1 :R= H
2 : R = COCH,CH,COOCH,
R = COCH,CH,COOH
Stmctures of Trp-P-2 and its haptenic derivatives.
in enzyme immunoassay. The maleimide derivatives widely used in immunoassay for bridging between the hapten molecule and carrier protein or enzyme are highly reactive to the thiol group . When compound 4 and the enzyme in molar ratios of 3 and 6 were subjected to preparation of the hapten-enzyme conjugate, 37 and 71% of the total enzyme activity were recovered in the immune-precipitate by the 1: 10 000 diluted antiserum. The optimal dilution of the antiserum was determined to be 1:20 000 for the enzyme conjugate of molar ratio 3 where 50% of the immunoreactive enzyme was precipitated. In this system the absorbance at 420 nm due to o-nitrophenol released by /3-galactosidase increased with incubation linearly up to 90 min. The newly established enzyme immunoassay system demonstrated a feasible dose-response
neutral fraction of pyrolyzed Amount
to the basic and
tar formed from tryptophan.
Trp-P-2 1 PS )
Fis. 2. Dose-response assay of Trp-P-2.
curve for the enzyme
curve for Trp-P-2 in the range of 20-800 pg per tube which was comparable to the ordinary sensitive radioimmunoassays (Fig. 2). It has previously been established in this laboratory that in the steroid enzyme immunoassay the bridge heterologous combination between the haptens used for the immunogen and labeled enzyme is favorable to develop the sensitive assay system [5,6]. This is, therefore, an additional instance demonstrating the validity of the proposed concept. In fact, it was unsuccessful to establish a sensitive assay system with the bridge
of the enzyme
homologous combination using the maleimide hapten (4) (data was not shown). The satisfactory recovery rate (97.9-109.6%) of Trp-P-2 added to the extract of pyrolyzed tryptophan justified the reliability of the values obtainable from the dose-response curve (Table I). The reproducibility of the enzyme immunoassay was also examined by the intra-and inter-assays with several different dilutions of the extract from tryptophan pyrolytic tar. As listed in Table II, the of this method proved to be precision satisfactory. The specificity of this enzyme immunoassay system was assessed by testing the crossreactivity to six related compounds (Table III). The l-ethyl derivative showed the highest crossreactivity (67%) among the compounds examin-
(n = 8)
(n = 5)
1:200 000 1:300 000
aOne milliliter of the maternal mg of L-tryptophan.
solution was equivalent
to the basic and neutral fraction of pyrolyzed
tar formed from 40
Table 111. Cross-reactivities
and amino acids in the enzyme
immunoassay. % Crossreactivity
R* CH3 H CH, H C,Hs
CH, CH, H
100 2.6 13 0.69 67
< 0.001 < 0.001
ed while 4-methylated (Trp-P-l) and I-demethylated derivatives showed only 13 and 2.6% respectively. Tryptophan and tryptamine, the precursors of y-carbolines in pyrolysis, showed no significant cross-reactivities. However, carcinogenic heteroaromatic compounds were isolated not only from the pyrolyzed tar of tryptophan but also from those of several other amino acids [ 131. The basic and neutral fraction of pyrolyzed tars formed from 12 amino acids and related compounds was, therefore, subjected to the enzyme immunoassay system (Table IV). It is noteworthy that 100 mg of 5-hydroxytryptophan produced the immunoreactive substance(s) equivalent to merely 8.3 pg of Trp-P-2 whereas an equal amount of tryptophan yielded 350 pg of Trp-P-2. This result implies the possibility whether this antibody would discriminate from the ring hydroxylated derivative or 5-hyroxytryptophan would be converted to y-carboline by pyrolysis in extremely low yield. It should be noted that 120 pg of TrpP-2 was determined in the pyrolyzed tar formed from tryptamine, which was comparable to that from tryptophan. Marked interference by immunoreactive substance(s) was observed in the pyrolyzed tar only from arginine among amino acids having no indole skeleton. As a preliminary study this assay system was applied to the basic fraction without the neutral com-
ponents from dried sardine prepared by the method of Yamaizumi et al. . In the fraction equivalent to 1 g of whole fish, Trp-P-2 was determined to be 60-80 ng (n = 4). This value is close in the order to that reported using GCMS . Although the Trp-P-2 level observed here in the pyrolyzed tar of tryptophan was
Table IV. Determination of immunoreactive substances to anti-trp-P-2 antibody in the basic and neutral fraction of pyrolyzed tar formed from various amino acids. Amino acid
Amount of immunoreactive substance (c(g Trp-P-2/100 mg amino acid)
L-Tryptophan Tryptamine HCI L-Arginine HCI L-5-Hydroxytryptophan L-Ornithine HCI Kitrulline l
r-Serine L.-Glutamic acid r-Histidine L.-Proline Creatine Creatinine N.D.: Not detectable.
350 120 10 8.3 1.2 1.1 0.68 0.48 0.33 N.D. N.D. N.D.
about 100 times higher than that reported by Kosuge et al. , the data described above led us to conclude that this enzyme immunoassay system is specific enough for evaluating the TrpP-2 level in the pyrolyzed amino acids and also possibly in broiled foods without tedious cleanup procedures. It is sufficiently substantiated that hydroxylation of the amino group on Trp-P-2 is the crucial step to form a covalent linkage to DNA molecule [3, lo]. Since the immunogen employed was attached to BSA through the amino group of the hapten, the antibody raised may be reactive to the adducts with nucleic acids and proteins. In this point of view the use of this antibody is of advantage not only for immunoassay but also for detecting the specific site damaged by TrpP-2 in vivo immunohistochemically.
(1981) Carcinogenic activity of 3-amino-1-methyl-5H-pyrido[4.3-b] indole (Trp-P-2), a pyrolysis product of tryptophan. 5
The authors express their sincere thanks to Dr. M. Okada and Prof. K. Shudo for generous gifts of the y-carboline derivatives. They are indebted to the staff of the central analytical laboratory of this Institute for elemental analyses and spectral measurements.
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