Indian Journal of Occupational and Environmental Medicine Medknow Publications ISSN: 0973-2284 EISSN: 1998-3670 Vol. 9, Num. 2, 2005, pp. 65-70

Indian Journal of Occupational and Environmental Medicine, Vol. 9, No. 2, May-August, 2005, pp. 65-70

Original Article

Vitellogenin assay by enzyme-linked immunosorbant assay as a biomarker of endocrine disruptor chemicals pollution

Ebrahimi M

Environmental Hormones Pollutants Research Center, University of Qom, Qom, Iran
Correspondence Address: Dr. M. Ebrahimi, Environmental Hormones Pollutants Research Center, University of QOM, QOM, Iran. ebrahimi@qom.ac.ir

Code Number: oe05016

Abstract

Keywords: ELISA, Endocrine disruptor chemicals, Immunoassay, Vitellogenin

Introduction

The last two decades have witnessed growing scientific concerns and public debate over the potential adverse effects that may result from exposure to a group of chemicals that have the potential to alter the normal functioning of the endocrine system in wildlife and humans.[1] According to WHO definition, an endocrine disruptor chemical (EDC) is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny or (sub) populations. The EDCs encompass a variety of chemical classes, including natural and synthetic hormones, plant constituents, pesticides, compounds used in the plastics industry and in consumer products, and other industrial by-products and pollutants.[2]

Mammalian studies have focused on the male since it is much easier to determine changes in the quality and quantity of sperm than of eggs, because eggs are few and are retained internally.[3] Fish have a major advantage over mammals in studies of endocrine disruptors in that they produce large numbers of both eggs and sperms and release them to the external environment. Hence, the effects of potential endocrine disruptors can be readily examined in both sexes. An assessment of pollution requires a simple biomarker and Vtg can be the easiest choice.

In very simple terms, vitellogenesis involves ovarian estradiol stimulating the liver to produce Vtg, which in turn is incorporated into the yolk of developing oocytes. Estrogenic xenobiotics can also act on the hepatic receptors to induce synthesis of Vtg.[4] Both male and female fish, as well as immature juveniles have hepatic estrogen receptors, but only the livers of female fish will normally be exposed to estrogens.[5] Production of Vtg by males and juveniles, or nonvitellogenic females can therefore provide a bioindicator of exposure to environmental estrogens.[6]

A number of studies have used Vtg to demonstrate the presence of estrogenic chemicals in the aquatic ecosystem. Trout exposed to sewage effluent in English rivers had up to 100 000-fold increase in plasma Vtg compared to fish upstream of the effluent.[7] A follow-up study in which caged trout were held in the River Aire, which is polluted by detergents from textile mills, showed Vtg levels of more than 20 mg/ml 5 km downstream of the effluent.[8] Elevated plasma Vtg was also found in eels below the Paris outfall.[9] Because eels may spend up to 20 years in the river before their spawning, migration-consequent reproductive disruption may account for some of the decrease in the European eel population.[9] The flounder, as a bottom-dwelling estuarine species, also provides a useful biomonitor of effluents in the rivers, which may potentially affect marine fish stocks.[10] In another study, male flounder in a number of British rivers had elevated plasma Vtg and, in the River Mersey, the concentrations were around 100 mg/ml (ten million times higher than controls).[11]

During the last 15 years, there have been a number of publications in which enzyme-linked immunosorbent assay (ELISA) using microtiter plates have been described as an alternative to radioimmunoassay for the mammalian and nonmammalian assays.[12] In addition to interest in measurement of steroids in fish plasma as an aid to aquaculture, there is increasing interest in such measurements to monitor the effects of endocrine disrupting chemicals on their reproductive function.[13] A report[14] has highlighted the need for a simple, rapid, and reliable method for the measurement of hormones specific to fish and other nonmammalian vertebrates as the basis for regulatory testing of the effects of potential endocrine disrupters on wildlife. In this communication, we describe a general preparation procedure for the enzyme label of carp Vtg, which is applicable to other species. The method was applied to the determination of Vtg production by ethynylestradiol, a potent xenoestrogen, in Carp ( Cyprinus carpio ) plasma.

Materials and Methods

Chemicals and equipment
The C. carpio Vtg and anti-Vtg antibodies were kind gifts from Professor Oliana Carnevali, Dipartimento di Biologia MCA, University of Camerino, 62032 Camerino, Italy. The purified Vtg is diluted to 10 and 50 mg/ml with Vtg storage buffer [PPBS, 0.1 M, pH 7.4; Potassium chloride, 27 mM; Tween-20, 0.05%; Bovine Serum Albumin, 0.2%; Sodium Azide, 5 mM; Aprotinin, 20 TIU/ml, 1% (Sigma, Deisenhofen, Germany)], divided into various volumes in eppendorf tubes and stored at -20°C. Goat F (ab′)2 fragment of affinity isolated rabbit antigen-specific antibody, conjugated to alkaline phosphatase was obtained from Sigma Immuno Chemicals (catalogue number A-3937). 4-Nitrophenylphosphate (pNPP) obtained from Boehringer Mannheim GmbH (catalogue number 107 905).

Ninety-six well polystyrene high-binding microtiter plates (Costar catalogue number 3590) were used for the ELISA. Plates were sealed for storage with Anachem Sealplate sealing film. Plates were processed using a Chelsea Instruments shaker, an Anthos Model AW-1 plate washer and read with an Anthos Model HTII Plate Reader. Ninety-six well low-binding microtiter plates (Costar catalogue number 2501) were used for sample dilution and preparation of standard curves and AnachemÒ SEALPLATE plate sealing film, (PK/100) purchased from Anachem (United Kingdom).

Fish exposure
Four groups of six male common carp (initial mean weight: 268.9 ± 52.5 g, mean ± SD) originating from a local fish breeder farm (Mahyar company, Iran) were kept in four separate tanks. They were exposed to 0, 5, 10, and 50 ng/ml ethynylestradiol for 30 days. Blood samples were taken at 0, 10, 20, and 30 days of exposure and plasma kept at -20°C until Vtg assay.

Plate coating with Vtg
ELISA plates were coated with 50 ng of purified Vtg. About 100 ml of carbonate buffer (0.05 M, pH 9.6) containing 500 ng/ml of purified standard Vtg added to each well of the plate and incubated for 1 h at room temperature in a humid container and then overnight at 4°C and washed once with nanopure water. The plate was blocked by the addition of a 200 ml per well of fresh Vtg blocking buffer [potassium phosphate buffer (PPB) 0.1 M, pH 7.4; bovine serum albumin, 2%; sodium azide, 5 mM], washed three times with wash buffer [Potassium phosphate buffered saline (PPBS), 0.02 M, pH 7.4; Tween-20, 0.05%] sealed with plate cover and stored at -20°C.

Standard assay procedure
Serial dilutions of purified standard Vtg (2000 - 3.9 ng/ml) in 100 ml Vtg assay buffer were made in the first ten wells of rows A and B of the coated plate. The remaining two wells in these rows were reserved for B0 (0pg) and nonspecific bound (NSB). Ten microliters of the diluted samples from sample dilution plate (2, 200, and 2000 times) was pipetted into the remaining six rows in duplicate. Twenty microliters of anti-Vtg antisera diluted 1/15 000 (carp) in Vtg assay buffer was added to all wells apart from NSB wells and made up the wells to 150 ml by Vtg assay buffer. The plates were sealed and incubated 2 h at room temperature in a humid chamber. Plates were then washed three times with wash buffer and 150 ml of anti-rabbit IgG alkaline phosphatase conjugate diluted 1/15 000 in Vtg assay buffer added to each well and incubated overnight at 4°C temperature. Plate was washed with wash buffer once and then twice with pNPP Substrate buffer (diethanolamine, 0.5 M, pH 9.8; MgCl, 0.5 mM), 175 ml/well of 1 mg/ml 4-nitrophenylphosphate in pNPP Substrate buffer added and then incubated in dark at room temperature until color developed to around 1.5 ODs (normally approximately 45 min but can be left up to 4 h). The reaction stopped by adding 50 ml of 3 M NaOH and read at 405 nm. Nanograms per ml were calculated for the samples from the standard curve using Stingray software (Dazdaq, Ringmer, UK).

Assay validation
A pool of male common carp ( C. carpio ) plasma (500 ml) was treated with 100°C for 1 h to remove all proteins from the plasma. One hundred microliter aliquots were pipetted in duplicate into wells on two rows of a microtiter plate. Pure Vtg was added to one pair of wells to give a concentration of 2000 ng/well, serial dilutions (2x) performed and the samples assayed according to standard procedure. The same procedure was used to assay Vtg in by carp exposed to ethynylestradiol (0, 5, 10, and 50 ng/ml). SPSS 10 for Windows software (SPSS Inc., 444 N. Michigan Avenue, Chicago, Illinois 60611, USA) was used for the statistical analysis of Vtg production in plasma of control fish group (0 ng/ml ethynylestradiol) with other experimental groups (5, 10, and 50 ng/ml ethynylestradiol).

Results

Standard curves and assay sensitivity
Typical standard curves for Vtg showed the working range of the ELISA was 11.25 - 2000 ng/ml [Figure - 1] [75 - 25% specific binding/maximum antibody binding (B/B0)] with a 50% B/B0 intra and interassay variation of 3.9% ( n = 10) [Figure - 2] and 12.5% ( n = 30), respectively. This ELISA is capable of detecting Vtg as low as 6 ng/ml.

Assay accuracy
There was a strong positive correlation ( r > 0.999) between the amount of Vtg added to deproteinated plasma and the amount found [Figure - 3].

Application to measuring Vtg production by carp exposed to ethynylestradiol
The Vtg was measured in duplicate by ELISA using 10 ml of the diluted plasma (2x, 200x, and 2000x) from fish exposed to different concentrations of ethynylestradiol. The amounts of Vtg detected by this method was 0, 402 ± 50.6, 6790 ± 1235, and 49824 ± 10423 mg/ml (mean ± standard error of mean, for six fish in each group) for fish exposed to 0, 5, 10, and 50 ng/ml of ethynylestradiol for 30 days respectively [Figure - 4]. A clear and potent estrogenic activity in inducing Vtg by fish liver was found with increase in ethynylestradiol concentration exposure and the amount of Vtg induced by different concentrations of ethynylestradiol between control (0 ng/ml) and experimental (5, 10, and 50 ng/ml) groups and between experiment groups were different significantly ( P < 0.05).

Discussion

Examination for the effects of potential EDCs begins only at levels at which there is no apparent stress or discomfort to the animal, so that it is potentially and equally able to reproduce.[15] Reproductive dysfunction at such low concentrations can be caused either by direct action on the gametes, or indirectly by modulation of the endocrine system so that gamete development takes place during an imbalance in the hormonal environment.[16]

In female fish, endocrine modulators can affect hypothalamic gonadotrophin releasing hormone (GnRH) secretion, pituitary gonadotrophin (GtH) release, estrogen biosynthesis and catabolism, estrogen binding to hepatic receptors, hepatic Vtg production, or the feedback by steroids and Vtg.[17] Although hepatic production of Vtg in male fish could be initiated by estrogens hormone, but no estrogenic hormones secrete in male fish and no Vtg production can be expected in normal life.[7] The use of in vitro fish hepatocyte cultures or in vivo fish exposure to estrogenic chemicals is shown to provide a simple and effective way to screen estrogenic activities of EDCs.[18] The Vtg assay in cell culture or fish plasma needs a quick, cheap, reliable, and efficient method to fulfill such a duty[19] and many researchers have already introduced different techniques to measure Vtg.[20],[7] An ELISA method for assaying Vtg has been described here.

Assay validation

Sensitivity: Sensitivity of the assays, which has been taken as 90% of the binding with 0pg is just over 10 mg for Vtg. Using identical conditions for label preparation and assay we also obtained a sensitivity of 2 mg for Vtg comparable to that obtained by others.[21] As with radioimmunoassay, the sensitivity is a function of both the specific activity of the label and the quality of the antiserum, and will therefore vary with the material available to the user. So in this study, we used the antiserum that were available to us but have little reason to expect that any antiserum which gives good results with RIA will not give similar results to those that we describe for ELISA.

Previous reports of ELISA for Vtg have used different enzyme labels[22] and in this communication a method described previously[23] and the results showed it is sufficiently sensitive for the rapid measurement of Vtg in common fish species such as cyprinids, and an example of such an application was described.

Accuracy and reproducibility: A major advantage of ELISA is the ability to minimize errors resulting from multiple pipetting by use of multichannel pipettes. Inter and intra-assay coefficient of variations (CV) reported in RIA are frequently 15-20% even when measured at the steepest part of the curve. For the assays reported here we obtain intra-assay CVs around 3.9% in the steep part of the curve, rising to 15-20% at the flatter end regions [Figure - 2], while interassay variation (at 50% displacement) was around 12%, comparable to that reported by Cuisset et al.[23] for 11-ketotestosterone steroid hormone. There was a highly significant correlation between the Vtg added to and that measured in deproteinized carp plasma [Figure - 3] and carp plasma. A further advantage of ELISA is that sample handling can also be made more rapid and reproducible by the use of low binding microtiter plates for sample storage and dispensing with multichannel pipettes

Application to the measurement of Vtg production in fish
To demonstrate an application of the ELISA method we have measured the amount of Vtg produced by carp exposed to ethynylestradiol. The results confirmed the previous findings that ethynylestradiol is a potent estrogenic chemical[24] [Figure - 4] and a clear, and potent estrogenic activity in inducing Vtg by fish was found with increase in ethynylestradiol concentration and the time of exposure (15 vs 30 days) [Figure - 4]. The amounts of Vtg production in fish exposed increased dramatically so that the logarithmic scales were used to demonstrate it as shown in [Figure - 4]. Such a potent induction of Vtg in other studies have already been discussed.[25]

The rapid determination of Vtg from the very small volumes of blood allows the examination of water pollution with EDCs and this methodology will therefore be of great interest to workers screening the estrogenic of new chemicals and phytoestrogens.

References

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