Immunochemical Approaches to Research on ... - ACS Publications

Chapter 15

Immunochemical Approaches to Research on Natural Toxicants and Phytoprotectants in Food

Downloaded by CORNELL UNIV on August 10, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch015

P. I. Creeke, H. A. Lee, M . R. A. Morgan, K. R. Price, M . J. C. Rhodes, and A. P. Wilkinson Department of Food Molecular Biochemistry, Institute of Food Research, Norwich Research Park, Colney, Norwich N R 4 7UA, United Kingdom

Immunochemical research on low molecular weight, biologically-active non-nutrient components of food materials has concentrated on contaminants such as mycotoxins and pesticides. Inherent components have attracted considerably less attention, despite evidence of considerably greater potential for in vivo bioactivity in man. Such activity might be deleterious (as is possible for the potato glycoalkaloids and the psoralens) or beneficial (such as the proposed protection against certain cancers provided by phytoestrogens like the isoflavones). Development and application of immunoassay methods of analysis offers advantages of specificity, a high rate of sample through-put and comparatively low cost. Particularly important is the potential high sensitivity. Application of immunoassays to human serum and tissues, where sample volumes are necessarily small, allows study of absorption and metabolism, and can result in improved assessments of implications for health. Examples of immunoassay approaches to the determination of the potato glycoalkaloids, isoflavones and psoralens will be presented. Immunoassay methods of analysis are now in wide use for detection and quantitative determination of low molecular weight contaminants of agri-food material. Indeed, a dedicated immunodiagnostics industry now provides all manner of antibody-based kits for a user community including the food producers, processors, retailers and consumers, as well as regulators and all those with environmental interests. Most of the attention has been directed towards contaminants such as the mycotoxins (including the aflatoxins, trichothecenes and fumonisins) and pesticides, in contrast, the natural bioactive, nonnutrient constituents of plant foods have received little attention. Plants are able to produce an extraordinarily diverse range of compounds through their pathways of secondary metabolism, many of them highly toxic to insects, fungii and other 'predators'. Indeed, the concept of 'natural pesticides' has been used to explain their activity and, possibly, their role in the plant. So poor is our basic knowledge of how the non-nutrient 0097-6156/96/0621-0202S15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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components of plant foods are absorbed and metabolized following ingestion, and what the effects are — i f any — on in vivo metabolism and health, that we can make few authoritative recommendations to consumers or plant breeders. This comment applies both to potentially deleterious and potentially beneficial plant non-nutrients. Food plants are rich sources of toxicants of wide-ranging activities, including many carcinogens. Despite this, virtually all available evidence points to the beneficial effects of consuming more fruit and vegetables, in terms of'protective' action against cancers and coronary heart disease. Such effects are related to the fibre and vitamin components, but there is a growing acceptance of the importance of the non-nutrient fraction (7). Immunoassay methods can readily provide information on dietary intake, absorption and metabolism. High levels of specificity and sensitivity coupled with high rates of sample through-put can be achieved. Specificity is needed because of the number of closely related compounds that might be present. Sensitivity is essential for several reasons; because of the low levels, because of the small sample volumes normally encountered in human studies in particular, and because sample work-up can be greatly simplified (eliminating purification steps) when it is possible to dilute the sample prior to analysis. The latter possibility also removes potentially interfering matrix effects. However, surprisingly few immunoassays have been developed for low molecular weight, non-nutrient components of plant foods (2). By far the most research has been devoted to the potato glycoalkaloids, toxicant steroidal alkaloids, and this work will be summarized in this and other chapters (See Sporns et al and Stanker et al, this volume). Of other toxicant plant secondary metabolites, the psoralens are, perhaps, of greatest interest because of their potent bioactivity, their high levels in certain plants, and the small safety margin that has been estimated between levels normally consumed and levels at which deleterious actions have been observed in humans. In this chapter we will further delineate reasons for conducting research involving immunoassay development for these compounds. As has been described previously, some of the non-nutrients are believed to provide beneficial health effects. Immunoassay can help elucidate the mechanisms involved, and lead to improved dietary advice. In this chapter we describe some of the immunochemical research carried out on the isoflavones, much of it related to deleterious effects in farm animals, but there is an increasing awareness because of the 'protective' effects that these compounds are thought to provide against certain human cancers. Psoralens. Psoralens (or linear furanocoumarins) are found in many commonly consumed vegetables (3) and fruits, including the plant families Rutaceae (citrus fruits), Umbelliferae (parsley, parsnip, celery, celeriac, fennel) and Moraceae (fig), and are thought to play an important role in protecting against attack by herbivores, insects and pathogens (4-9). A large number of psoralens have been isolated from plants, the most common are shown in Figure 1. Psoralen is the biosynthetic precursor (9) for the other psoralens and is elaborated by hydoxylation and O-alkylation. The levels of psoralens found in commonly consumed vegetables (Table I) (70) are variable and can be considerably higher in stressed plants (11,12). On the surface of spoiled parsnips, levels of xanthotoxin, psoralen and bergapten of 110.9 mg/100 g, 53.7 mg/100 g and 9.0 mg/100 g, respectively, have been found and these levels persisted in apparently healthy tissue below the diseased surface (73).

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


204

IMMUNOASSAYS FOR RESIDUE ANALYSIS

Common name

Ri

R

Psoralen

H

H

Xanthotoxin

H

OMe

Bergapten

OMe

H

Isopimpinellin

OMe

OMe

Isoimperatorin

OCH CH=C(CH )

Oxypeucedanin

OCH CH-C(CH )

2

2

Heraclenin Oxypeucedanin hydrate Figure 1.

3

H

OCH CH-^C 2

OCH CHOHCOH(CH ) 2

H

2

3

H

2

3

2

H

Structures of commonly occurring plant psoralens.


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Table L Levels of psoralens in healthy vegetables (mg/100 g). Bergapten

Isopimpinellin

Psoralen

Xanthotoxin

Celeriac

0-3.15

0-1.26

0-1.03

0.12-2.16

Celery

0-1.85

0-2.05

0-1.24

0-0.82

Parsley

0.10-1.82

0 - 0.22

0-0.31

0-0.45

Parsnip

0.22 - 0.80

0.10-1.36

0.26-5.45


Vegetable

Levels of psoralens vary considerable from one part of the plant to another, in general the concentrations are highest in the outer leaves (14), and peel. When the peel is used, as in the production of essential oils from citrus fruit, the levels of psoralens can be very high (15), for example bergamot oil can contain up to 0.8% bergapten (16). Pharmacology. In combination with U V light, many psoralens are potent photosentisizers, and the consumption of psoralens has resulted in phototoxic reactions in man and animals (17). The psoralens are known to cause mutagenic and lethal effects in vitro and in vivo (18). Understanding of the biological actions of psoralens has come primarily through interest in their pharmacological properties. The therapeutic benefits of psoralens and light have been known from ancient times (79). Psoralens, commonly bergapten, xanthotoxin, and 4,5,8-trimethylpsoralen in combination with U V A light (320-400 nm), are used clinically in the treatment of a range of skin disorders including psoriasis (20), vitiligo (27), mycosis fungoides (22), and leukaemic cutaneous T-cell lymphomas (23). Psoralens have been used in a number of sun-tan preparations as a stimulus to the production of melanin (18). In addition, psoralens show anti-depressant activity (24) (possibly via the stimulation of the production of melatonin), are effective potassium channel blockers with a possible role in treating demyelinating diseases like multiple sclerosis (25), and show cytostatic activity (26). Psoralens also have a number of overtly deleterious actions (27); they interact with D N A , are activated by cytochrome P-450 enzymes, and cause photosentization reactions. D N A Adduct Formation. Psoralens intercalate into the minor groove of D N A duplexes, with the 5'-TA sequence as the preferred site (28), and in combination with U V light, undergo a [2+2] cycloaddition (29) reaction with the pyrimidine bases (primarily thymine) forming both monofiinctional and bifunctional (cross linking) adducts (27). In vitro and in vivo studies have shown these photoadducts to be mutagenic and carcinogenic. In addition, psoralens have been shown to be mutagenic in the absence of light (30). Studies in humans suggest that patients who have received psoralen treatment are at a ten times greater risk of developing cutaneous squamous-cell carcinoma (31,32). The World Health Organization has cited psoralen phototherapy as a cause of human skin cancer (33).


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Interaction with Cytochrome P-450. Psoralens are potent modulators of cytochrome P-450 (34,35,36). Xanthotoxin has been shown to be biotransformed in vitro to reactive intermediates (possible intermediates are a furan epoxide and an unsaturated dicarbonyl compound) that bind covalently to liver microsomes. These activated species react with a wide range of nucleophilic groups including D N A , R N A and proteins. It should be noted that anatoxin is activated in a similar manner, to give a 2,3-epoxy-aflatoxin, and is known to be a potent carcinogen causing cancer of the liver, colon, and kidney in some animals (37). Psoralens also deactivate certain Cytochrome P-450 enzymes affecting the metabolism of xenobiotics. The mechanism of inactivation of cytochrome PASO is not clear, but might result from covalent binding of activated xanthotoxin intermediates to specific target cytochrome P-450 (35). Photosentisation. The precise mechanism by which psoralens cause skin photosensitization reactions is not known (38). Some of the biological effects of psoralens in the skin have been attributed to the formation of adducts with DNA. However an alternative mechanism has been proposed involving the binding of psoralens to specific receptors in cell membranes and cytoplasm that are important in the regulation of growth factor-induced signal transduction pathways. Following U V irradiation the receptor is activated by photoalkylation, leading to a cascade of biochemical changes that result in alterations in cell growth and differentiation. Human Exposure. Humans are exposed to psoralens through handling or ingesting psoralen containing fruits and vegetables. High levels have been associated with outbreaks of photodermatitis in workers handling produce, especially parsnips, celery (14,39), citrus fruit (40), and the use of fragrances. A number of cases have been documented where severe phytophotodermatitis has occurred following consumption of limes (40), and celery (41,42,43). In addition, cataract (pigmented spots) formation is a possible risk associated with the consumption of psoralens, and has been noted in farm animals (17), rats (44), and humans (45). Studies in humans show that the phototoxic threshold of an ingested dose of a mixture of bergapten and xanthotoxin is about 10 mg xanthotoxin and 10 mg of bergapten, and for xanthotoxin alone about 15 mg. This dose is not normally reached by the consumption of healthy vegetables under normal dietary habits, however the estimated safety factor is relatively small, about 2-10 pg/g (46). Concentrations of psoralens of 8-10 pg/g celery were found to cause phytodermatitis in grocery store workers (40). Metabolism of Bergapten and Xanthotoxin in Man. Bergapten and xanthotoxin are extensively metabolized in man (47-49), and are rapidly cleared, with serum half-lives of about 1 h (50). The major metabolites of bergapten and xanthotoxin are glucuronic acid conjugates, mainly of arylacetic acid and arylalcohols resulting from oxidation of the furan ring, possible via an intermediate 2',3'-epoxide. Reduction of the 3,4-double bond in the lactone ring of bergapten, followed by hydrolysis to give an arylpropionic acid derivative, is thought to result from metabolism by gastrointestinal microflora. Metabolism of the lactone ring of xanthotoxin occurs to a minor extent yielding an arylpropenoic acid derivative.



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Development of E L I S A Methods for Psoralens. No work has been reported in the literature concerning the development of immunoassays for the analysis of free psoralens of food origin. However, ELISA methods have been developed for DNA-psoralen adducts (31,51,52), and applied to the quantification of D N A adducts in plasma from patients undergoing psoralen treatment, and to human keratinocytes and animal skin samples. No work was done on the quantification of adducts arising from the exposure to, or consumption of psoralens in the diet; such work might give an indication as to what the long term risks are. The four most commonly found psoralens (psoralen, bergapten, xanthotoxin, and isopimpinellin) in fruits and vegetables are currently the subject of immunoassay development in this laboratory. The aim of this work will be to explore the dietary absorption and bioavailability of these compounds with a view to further elucidate the real risks (if any) of dietary psoralen consumption. Antisera against xanthotoxol and xanthotoxin have been produced using xanthotoxin immunogens shown in Figures 2 and 3 (Creeke, Lee, Wilkinson and Morgan, Institute of Food Research at Norwich, U K , unpublished data) and are currently being assessed for suitability for plant and human studies. It is hoped that further understanding of the role of psoralens in human health will be forthcoming. Isoflavones. Isoflavones are heterocyclic diphenols. More than 200 types have been described principally in plants of the Leguminosae sub-family (53) some of which are plants regularly consumed by humans such as soya (Glycine max) and chickpea (Cicer arietinum) (54). Of the 200 different types, a small number have been shown to be estrogenic in animals. Interest in the estrogenic activity of plant isoflavones stems from the 1940s when they were shown to be responsible for 'clover disease', an infertility syndrome in sheep that devastated the Western Australian sheep breeding industry (55). The affected sheep had fed on subterranean clover (Trifolium subterraneum) which had levels of up to 5% dry matter (56) of isoflavones consisting of formononetin, biochanin A, genistein and daidzein. The latter pair occurring as 7-O-glycosides (genistin and daidzin; Figure 4). Subsequently, isoflavones have been shown to be estrogenic in a number of animal species such as cattle (57), rats (58), mice (56), Californien quails (60) and cheetahs (61). The relative estrogenic activity of individual isoflavones varies with animal species and also route of administration (62,63). The most estrogenic all have a spatial arrangement of functional groups, particularly phenolic groups, similar to natural and synthetic estrogens (64) (Figure 4). Isoflavones and Human Health. The estrogenicity of isoflavones is weak in comparison with natural estrogens such as estradiol (Table Π) and as a result they were not considered to be a health problem for humans. However, over the last 10 years increasing interest has been shown in dietary isoflavones as a growing body of evidence suggests that they may be beneficial to human health. Epidemiological data shows that in Asian countries there is a much lower incidence of certain cancers such as breast and prostate, than in western countries. This difference has been attributed to the much greater use of soya in Asian diets (64,66-72). Soya foods can contain up to 3% by weight of isoflavones, mainly the glycosides daidzin and


208



1. m-CPBA

Figure 2.

Synthesis of xanthotoxol-protein conjugates. (m-CPBA = 3-chloroperoxybenzoic acid; FMPTS = 2-fluoro-l-methylpyridinium /7-toluenesulfonate; CDI = Ι,Γ-carbonyl-diimidazole).

Figure 3.

Synthesis of xanthotoxin-protein conjugate.


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genistin (73). Endogenous and synthetic hormones are important in the development of a number of hormone dependent cancers, including cancer of the breast and prostate. It is thought that because of their low estrogenicity, isoflavones can antagonize hormonedependent cancer development through binding estrogen receptors, or by altering sex hormone metabolism, in a manner that reduces the total effective life-time exposure to endogenous estrogens (64,70,72). In addition, isoflavones have the potential to protect against cancer in a non-steroidal manner because they are antioxidants, and can inhibit cell proliferation, tumour induction and angiogenesis (64,70).


Table Π. Relative estrogenicity of phytoestrogens to estradiol upon a human endometrial cell line. Compound Estradiol

Relative Estrogenicity 100

Genistein

0.08

Equol

0.06

Daidzein

0.01

Biochanin A

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