FEATURE

Monitoring Endocrine-Disrupting Chemicals OMOWUNMI A. SADIK AND DIANE M. WITT

Novel strategies

are providing a more

comprehensive and

refined basis for

understanding the

occurrence and

effects of endocrine

disrupters.

The effect of endocrine-disrupting chemicals (EDCs) on human health and wildlife is re­ceiving growing attention from the scien­tific community, regulatory agencies, and the public at large. Exposure to EDCs (see Table

1) can mimic or interfere with mechanisms of nat­urally occurring hormones responsible for regulat­ing reproductive and developmental bodily pro­cesses of the body (1-3). The chemicals can also dramatically alter normal physiological function­ing of the endocrine system and can affect forma­tion (synthesis) of hormones in the body or target tissues where the hormones exert their effects.

Driven by this increasing awareness of possible EDC impacts, a recent government-wide effort is stimulating innovative, multidisciplinary research that addresses scientific uncertainties concerning poten­tial adverse effects of the chemicals. The collabora­tive effort involves experts from EPA, the U.S. De­partment of Interior, the National Institute of Environmental Health Sciences, National Oceanic and Atmospheric Administration, the U.S. Department of Agriculture, the U.S. Department of Energy, the Cen­ters for Disease Control and Prevention, and the U.S. Food and Drug Administration.

Undertakings in the areas of human health, eco­logical effects, and exposure assessment are consid­ered priority research needs essential to the larger goal of determining the extent and magnitude of endo­crine distrupter impacts. The most critical step to­ward achieving this goal is to determine which pollut­ants can definitely be characterized as environmental endocrine disrupters.

Unfortunately, although endocrine-disrupting chemicals are of great interest to agencies respon­sible for determining their potential effects on hu­mans and wildlife, currently, no suitable character­ization method exists for evaluating most EDCs in

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biota or biological media. Moreover, monitoring and surveillance studies of EDCs are difficult undertak­ings. In addition, screening (or testing) of the wide variety of syntiietic EDCs and their metabolites in­volves substantial costs and time.

Adding to these complexities is that, unlike most toxic substances, EDCs generally exhibit unusual "dose-response" behaviors, thus rendering the thresh­old concept of toxicology inapplicable for EDC stud­ies. The dose-response behaviors for most toxic sub­stances usually increase with increasing levels of chemical compounds, which eventually level off. In contrast, the response curves for environmental es­trogens exhibit an inverted U-shape—the greatest re­sponse is produced at extremely low doses.

There are also concerns that the effects of differ­ent EDCs are additive, or even synergistic, such that regulating individual compounds may be inade­quate. Moreover, the process of enabling estro­genic activities in some tissues, while completely blocking these activities in others, could result in an increase in estrogenic activities of certain EDCs found in tissues. This phenomenon is known as an "ago­nistic and antagonistic" effect.

Issues such as these are being addressed through the development of novel monitoring strategies that involve use of bioaffinity sensors, cell-based as­says, acute slice explants, and hyphenated tech­niques. The monitoring strategies can be based on combinations of short-term screening, high-performance testing, long-term behavioral testing, and animal testing. Such approaches seek to capi­talize on advantages of and surmount limitations of existing analytical techniques. Taken together, these approaches should provide viable strategies for char­acterizing potential environmental estrogens.

Exposure and impact Exposure to EDCs can occur during pregnancy through the transfer of blood-borne agents from the mother to the fetus, which can cause hormonal im­balance or changes in receptor sensitivities in the fe­tus, ending in reproductive abnormalities. It is al­ready well established mat environmental factors can directly influence genes and physiological mecha­nisms underlying sexual differentiation of brain struc­tures, genitalia, and behavioral characteristics.

Prenatal or postnatal exposure to EDCs can per-manendy alter the neuroendocrine properties of the central nervous system, interfering with the feedback systems that are normal features of the pituitary, go­nads, adrenal, and thyroid axes in males and females. EDCs may also indirectly affect plasma steroid levels by altering hormonal activities in the gonads or adre­nals or by modifying die second messenger systems in the brain. For example, polychlorinated biphenyls (PCBs) appear to interfere with gonadotropin secre­tion because of changes in neurotransmitter activity in the hypothalamus (4).

Many EDCs compete with estradiol for the estro­gen receptor, whereas omers compete with dihydrotes-tosterone for the androgen receptor (5). Therefore, EDCs are capable of altering the endocrine system by affect­ing hormone synthesis or degradation, transport, re­ceptor binding properties, and gene transcription. EDCs may even eliminate the natural-borne hormones re­sponsible for regulating homeostasis, as well as devel­opmental and reproductive processes {1, 2).

Testing and surveillance requirements Prompted by this new awareness of potential im­pacts of EDCs, scientists are looking for efficient, low-cost screening strategies capable of identifying and

TABLE 1

Selected endocrine-disrupting chemicals

Endocrine-disrupting chemicals, most of which are present at parts-per-billion levels, include compounds such as bioaccumulative

organochlorines, pesticides, industrial chemicals, and phytoestrogens—naturally occurring EDCs.

Group 1 Chlorinated organics

2,4,5-Trichloro-phenol (S)

PCBs (K)

2,3,7,8-TCDD (K)

Pentachlorophenol (K)

Group 2 Industrial chemicals

Hydrazine* (P)

Bisphenol A (P) Transplatin (S) Benzoflumethiazide Propantheline-bromide (S)

Butyl benzyl pthalate (S)

p-Nitrotoluene

Obidoxime chloride (S)

uiuup o Polymers with molecular weights 1000 or less

Fluoropolyol

Methoxy-polysiloxane (P)

Polyethylene oxide (S)

Poly(isobutylene)

Polyurethane (S)

Group 4 TSCA

PAHs (K)

Triazines (K) Kepone(S)

Lead (K)

Mercury (K)

Endosulfans

Group 5 Pesticides

Malathion (S)

1,2-Dibromo-3-chloropropane Aldicarb

nitrofen (S)

Pyrethroids (S)

Dicamba (S)

Aldicarb (P)

* Currently no available EPA method. P denotes probable EDC; S denotes suspected EDC; K denotes known EDC

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classifying all EDCs, as well as potentially contam­inated sites within the environment. Once these screening methods are developed, further labora­tory reanalysis, including confirmation and long-term studies, will be necessary to identify endocrine-disrupting characteristics among the population.

The amount of screening that will be required is enormous, given the wide range of EDCs, metabo­lites, and potential environmental estrogens. Accord­ing to a recent EPA advisory committee on endo­crine disrupters, several commercial chemicals, including nearly 87,000 mixtures, should be screened to assess and determine their effects on the endo­crine system (6). The relative potencies of synthetic and natural EDCs include highly target, organ-specific species, and the differences could compli­cate hazard assessment (7).

Development and deployment of screening tech­niques are important prerequisites for determining the behavioral effects of EDCs, an area that has been largely neglected by researchers. Understanding both the phar macokinetics and mechanisms of action, while local­izing the specific effects of EDCs on target tissue, may be equally vital for developing biochemical interven­tions capable of diminishing the impact of EDCs.

Most important, such studies should generate the detailed information needed to avoid synthesizing sim­ilar compounds that could have ill effects on the en­docrine system. This task will not be easy. The un­usual dose-response behavior of EDCs complicates testing and surveillance: Results obtained for both es­tradiol and diethylstilbestrol in prostrate indicated a re­sponse that first decreased with dose, then increased, producing the inverted U-shaped dose-response re­lationship (8). Moreover, the use of threshold infer­ence is difficult in the case of many xenobiotics, be­cause these compounds readily mimic the endogenous actions of steroidal compounds essential for development. Thus, the threshold is automatically ex­ceeded with continued exposure to these substances.

Also posing a challenge for monitoring is that the effects of different EDCs can be additive, or even syn­ergistic. A typical range of synergistic endocrine re­ceptor binding, which includes the induction of re­porter gene activity compared with that observed for the compounds alone, is between 160- and 1600-fold. In one yeast-based assay, the binary mixtures of weak estrogenic pesticides, including dieldrin, chlordane, toxaphene, and endosulfan, exhibited greater than a 90-fold effect (9).

TABLE 2

Current monitoring methods for endocrine-disrupting chemicals There are specific advantages and disadvantages of techniques used for characterizing endocrine-disrupting chemicals.

Characterization method

Bioassays

Receptor-binding assays

DNA-binding assays

Cell-based assays

Chromatographic methods

Principle

Quantitation of the response fol lowing the application of a stimulus to a biological system

Principle similar to immunoassays in which antibody is replaced with

enzyme-linked estrogen receptor

DNA binding of estrogen receptor by suitable ligands

Proliferation of cells on incubation with estrogenic compounds

Separating components of materials between two immiscible phases on the basis of differences in their molecular size, charge, mass, polarities, and redox potentials.

Advantages

Similar to living situation/ incorporates the effects of metabolism, serum binding,

and pharmacokinetics

Useful for pharmacolo­gically and toxicologically relevant substances in

unknown samples; adaptable to f low injection analysis mode; easy to perform; allows identifi­cation; rapid analysis t ime

Suitable for studying important biological effects of active compounds through the cell membrane

Suitable for primary identi­fication of estrogenic activities of several compounds

Structure confirmation and analysis of estrogenic compounds (particularly

GC/MS)

Disadvantages 1

Large-scale screening requires faster and less expensive alternatives.

Unable to differentiate the binding of agonist and antagonist EDCs

May be unsuitable for the detection of synergistic effects of natural and environmental estrogens

Length of assays is about 4-6 days, making it less attractive for large-scale screening.

Potential loss in specificity and high cost; GC/MS requires derivatization of the estrogenic compounds in order to be volatile; Low levels in biological matrices make quantitation using UV-vis difficult.

Reference

[7,8)

(6)

(6)

3 7 0 A • SEPTEMBER 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS

Current monitoring techniques Most studies involving the analysis of EDCs have focused on estrogens using bioassay techniques. Bioassay involves the quantitation of a biolog­ical response that follows the appli­cation of a stimulus to a living or­ganism. The quantitative response can be seen in some aspects of the biological system, producing a pos­itive or negative signal such as an in­creased activity, a negative response (inhibition), or even death to the bi­ological system. The response ob­tained provides information on the biological activity that is normally at­tributed to the analyte.

One major advantage of the bio­assay is its specificity. Specificity is particularly attractive for any char­acterization in which the mixture of agonist and antagonist forms is present but cannot be separated ef­fectively. Bioassays are often very sensitive and can distinguish very small differences in analyte activi­ties, particularly when insufficient material is available. The use of an­imal bioassays is becoming less fre­quent because of improvements in both the purification and alterna­tive analytical techniques and be­cause of the advent of sophisti­cated tools in molecular biology for detecting and accurately measur­ing biological products. In addi­tion, large-scale screening using bio­assays requires faster and less expensive alternatives.

Other modifications of bioassays for in vitro analyses commonly used for the characterization of EDCs in­clude receptor-binding assays, bio­sensors, DNA-binding assays, cell-based assays and recombinant yeast-screen assays (see Table 2). For example, enzyme-linked receptor as­says (ELRAs) have been used to in­vestigate estrogenic activities in vitro involving estrogen receptors (6). Ex­periments conducted on 17|}-estra-diol resulted in a detection limit of 0.1 ng/mL. Although an ELRA is sim­ple to perform (and can identify all of the EDCs that act through estro­gen receptors), the major limitation lies in its inability to differentiate the functional binding activities of ago­nist or antagonist EDCs.

Well-known synthetic EDCs are generally assayed using conven­tional analytical techniques, includ­ing liquid chromatography, gas chromatography, and hyphenated

FIGURE 1

Biosensor surface preparation Biosensors can be designed with predefined functionality for the analysis of specific endocrine-disrupting chemicals (EDCs). The sensor consists of an artificial interface located on the surface of a transducer. This interface can be engineered to have a predefined affinity for target analytes (such as EDCs). Sensor preparation requires immobilization of two types of molecules. The first molecule serves as a "template," having a shape similar to the analyte of interest and contains an additional functional group that is capable of strongly binding with the transducer surface. The second molecule acts as a "substrate" capable of strong adsorption on the surface and forms a monolayer thicker than itself. Because of coadsorption of the two molecules on the transducer surface, a mixed monolayer matrix, consisting of template and substrate, forms. This resulting monolayer contains template-shaped cavities that do not undergo lateral diffusion and exhibit a pronounced affinity for the analyte molecule of interest. Such predefined affinity interfaces can also be prepared by techniques other than adsorption, for example, covalent binding, cross-linking, entrapment, or microencapsulation.

SEPTEMBER 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 3 7 1 A

FIGURE 2

Operation of a whole-cell biosensor Bacterial cells used in this application are grown on a single carbon or nitrogen source. These cells, thus grown, contain a very large amount of a particular enzyme (significant for its selectivity). The presence of analytes (such as endocrine-disrupting chemicals (EDCs)) often leads to substantial increases in the metabolic activities that cause production of electroactive species generating as a result, a large potentiometric signal. Mediator molecules facilitate the transfer of electrons from the cells to the surface of the indicator electrode, by interacting at certain sites along the electron transfer chain. The effect of this interaction causes the mediators to become reduced as the chain progresses—this causes the me­diator molecules to act like termi­nal electron acceptors. Subse­quent reoxidation of the reduced mediator molecules at the work­ing electrode results in a steady flow of current that can be mea­sured by an external circuitry. The magnitude of this current is proportional to the activity of the bacterial organisms, so that any perturbation in the ensuing cur­rent time curve indicates the presence of an EDC pollutant.

techniques such as gas chromatography and mass spectrometry (GC/MS). The advantages of these tech­niques include their accuracy and precision. Gas chromatography (GC) and liquid chromatography (LC) techniques are used generally for structure con­firmation and the validation of environmental sam­ples. A wide range of available analytical methods can be used to analyze other known EDCs, such as those listed in Table 1.

These techniques have known limitations. Us­ing GC-based techniques for analysis of potential EDCs requires derivatization to increase their vola­tility prior to mass spectroscopic identification. LC methods require the use of ultrasensitive detectors for monitoring dynamic events and low levels of EDCs usually found in biological matrices. These tech­niques are costly, generally have low analytical throughputs, and require more complex instrumen­tation. Extensive validation studies are required to eliminate interference problems.

Multidimensional GCs, including fast GCs and high-resolution MS, are capable of overcoming these limitations. However, for biomonitoring purposes, in particular, the analysis of EDCs, the potential result­ing loss in specificity (such as coelution of peaks), and issues of resolution and sensitivity must be ad­dressed. To ascertain estrogenic activities of a com­pound using classical chromatographic tech­niques, these methods must be combined with bioassays or other receptor-based techniques. For these reasons, researchers are looking at the rele­vance of existing methodologies in providing unam­

biguous analysis results, or in some cases, are sim­ply seeking new approaches for the characterization of potential EDCs.

Novel monitoring methods Sophisticated biosensors are used for rapid and re­liable measurement of harmful substances and bio­chemical compounds, including triazines, phenols, polyaromatic hydrocarbons, and others that are of military interest or are relevant to the food indus­try. The evolution of tiiese novel sensors has brought about a growth in environmental measurement tech­nique capabilities and has accelerated understand­ing of biochemical reactions.

A typical biosensor consists of a biological-sensing element (such as an antibody), whole cells, and receptors, which are in close proximity to a transducer (such as an electrode, an optical fiber, or a piezoelectric or mass transducer). The biolog­ical element may be connected directly to or inte­grated within the transducer. Measurement of a tar­get analyte (such as an EDC) can be achieved by selectively converting the molecular recognition oc­curring at the analyte-sensor interface from a non­electrical domain to an electrical signal.

Several bioaffinity reagents can be used to con­struct a sensor, including receptors, binding pro­teins, and nucleic acids immobilized on the surface of the transducer. It is possible to couple the recep­tor to the sensor surface using an antibody that can recognize the binding site distinctly from the active site. Care must be taken to ensure that the anti­body binding causes no conformational change in the receptor in a way that could alter the function of the active site. Once the purified receptor has been coupled to the sensor surface, the binding of the EDCs can be detected through a change in the phys­ical property at the sensing surface.

In our laboratories the renewable immunosen-sors and electrochemical microassembly technolo­gies have been used to analyze a range of sus­pected EDCs, including PCBs, chlorinated phenols, atrazines, and heavy metals [10-12).

In bioaffinity sensors, the ability of EDCs to in­terfere with the hormone system is a major factor used to develop a (rapid) detection method. The ar­chitecture of these sensors is similar to those com­monly used in immunoassays. In this case, the en­docrine disrupter of interest is permitted to compete with and displace a fluorescent (or enzyme-labeled) receptor. The quantity of the endocrine dis­rupter is then determined using the standard curve obtained from the quantity of the fluorescent mol­ecule remaining after the system reaches equilib­rium, which requires only 1-3 minutes.

Bioaffinity sensors provide two ways of differen­tiating target from interfering compounds. The first is associated with construction of the sensing de­vice—differentiation is realized by preparing an ar­tificial template with a predefined affinity. A sec­ond level of discrimination is realized through the use of relative binding affinities. These sensors (see Figure 1) are capable of separating an individual com­ponent or a selected range of components from com-

3 7 2 A • SEPTEMBER 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS

plex mixtures of biomolecules (on the basis of their chemical structures and biological functions).

New monitoring strategies On the basis of EPA's advisory committee's testing re­gime for various chemicals (see Table 1), a strategy based on three levels of screening has been recom­mended for addressing the wide range of possible EDCs, metabolites, and potential environmental estrogens.

Prescreening of EDCs using biological assays. Bi­ological assays such as ELRAs, DNA-binding as­says, and biosensors that can provide rapid "yes or no" answers should first be carried out, preferably on site. This step can eliminate chemicals that do not exhibit endocrine characteristics. For example, af­finity biosensors can be used to successfully screen chemical compounds provided a suitable antibody or receptor is available. Alternatively, cells can be grown in an appropriate medium for in situ screen­ing of EDCs. The performance of these sensors is di­rectly linked to their sensitivity, limit of detection, pre­cision, and accuracy. The most important of these factors is sensor specificity, including minimal in­terference from other structurally similar com­pounds and metabolites. If the screening studies re­sult in any significant amount of interference, the chemical can be classified as an endocrine dis­rupter and subsequently assigned a quantitative en­docrine-disrupting index based on a combination of its IC50 values (a measure of incapacitation effect), rate constants, and known chemical structure.

Multidimensional bioassay, chromatographic, and flow injection analysis techniques. The next level of screening should involve methods that use renew­able surfaces for rapid, high-performance, and on­line screening assays. This can further test the abil­ity of a chemical to attach to a nominated receptor and provide information derived from cultured cells. Affinity chromatography, high-throughput GCs, flow injection analysis, two-dimensional GCs, high-resolution MS, and LC techniques are examples of analytical methods that could be integrated with bi­osensors to provide this second level of EDC screen­ing. These methods provide the necessary selectiv­ity, as well as continuous monitoring capabilities. Suitable detectors include arrays of cell-based sen­sors and receptor-based assays that incorporate a net­work of intact biological cells as the detection-measurement elements.

Cell-based biosensors can provide alternative tem­plates for screening EDCs because animals are not suitable for continuous monitoring, quantitative anal­ysis, or any screening in hostile environments. Cell biosensors are also suitable for studying the cellu­lar (and molecular) mechanism of interactions be­tween EDCs and steroids of the central nervous sys­tem. By responding to a wide range of chemicals through biological reaction sequences, cells can be used to promote optimum analyte-receptor activi­ties. Such sensors can provide a low-cost alterna­tive for in situ monitoring of EDCs. In whole cell-based assays, indicator electrodes are used in conjunction with immobilized bacteria entrapped in a gel or membrane support (see Figure 2). In con­

structing these biosensors, the cells are immobi­lized onto the surface of bacteriological filters that allow the diffusion of the toxicant (such as an EDC) and the mediator molecules to the cells, as well as the diffusion of the reduced mediator molecules to the electrode.

Immunocytochemical methods and long-term an­imal studies. If and when positive or equivocal re­sults are obtained in any of the above regimes, those chemicals should undergo further detailed in vitro analyses of the effects of EDCs on the central ner­vous system prior to long-term animal studies. An example of suitable techniques includes immuno­cytochemical methods for localizing the brain re­gions responsive to the tested EDCs. Another method is simulated in situ histochemistry for measuring the alterations in receptor messenger RNA. To charac­terize the direct effects of EDCs on the physiology of an organism, we must specifically determine how the EDCs "reorganize" neural substrates underlying reproductive physiology and behavior. The determi­nation of the specific neural mechanisms involved in EDC exposure is clearly limited by the complex­ities of the in vivo environment in the brain, which consists of a multitude of neural networks and feed­back circuits.

The specific cellular responses following EDC ex­posure are particularly difficult to analyze in vivo, and such phenomena should be examined initially in vitro, under highly controlled conditions. We have devel­oped a screening technique to isolate and identify in­tact neuroanatomic structures that might be highly re­sponsive to EDCs. Information derived from these studies will be instrumental in identifying specific brain regions most often affected by EDC exposure and de­termining cellular phenotypes that might be directly involved in EDC-mediated physiological and behav­ioral responses. These data will delineate which brain areas should be the primary focus of subsequent stud­ies and will eliminate wasted time and effort on sys­tematic in vivo searching for a myriad of neural struc­tures that may respond to EDCs.

Future research areas In vitro studies of EDC actions in humans and wild­life. An in vitro system provides the opportunity to extensively characterize the effects of EDCs in spe­cific brain nuclei. Current in vitro models allow pre­cision in timing, concentrations, and length of ex­posure, but they also have their limitations. The primary culture of brain cells, for long-period in vitro, often alters normal neuronal properties. Typically, the mechanisms affecting receptor synthesis or activa­tion are studied in homogenate preparations of pri­mary dissociated neurons or cultures of trans­formed cell lines. In such preparations, the fundamental neuroanatomic specificity and synap­tic organization are absent, and therefore many of the regulatory mechanisms that operate within the interneuronal circuits in vivo may not be ade­quately represented.

To circumvent some of the problems usually as­sociated with long-term in vitro tissue culture, fu­ture areas of research must involve the develop-

SEPTEMBER 1, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 3 7 3 A

ment of viable alternatives that can provide important insights into the in vivo effects of EDCs on gene ex­pression. For example, we have made a novel adap­tation to an existing slice explant technology typi­cally used for electrophysiological studies and developed an in vitro method to study gene expres­sion, referred to as the acute slice explants (ASE) tech­nique {13, 14).

Quantitative structure-activity relationships (QSAR). Research in this area is expected to deepen understanding of the nature of EDC actions in hu­mans and wildlife. QSAR represents an empirical way of connecting the functional binding affinity of a se­ries of structurally related EDCs to its endocrine-disrupting action. Simple QSAR relationships may ex­ist between the binding energies of an EDC molecule and estradiol. Other important parameters may in­clude hydrophobicity, binding constant, d o s e -response behaviors, and polarity of the chemical groups. The QSAR concept may be refined by as­signing empirical values to structural features of a molecule such as size, shape, electron distribution, and hydrogen bond capability. Multiple linear re­gression should produce a quantitative structure-activity relationship; the magnitudes of which coef­ficient and regression parameters could reveal which structural features are important in determining the endocrine-disrupting characteristics. This informa­tion could be extracted from the three-level moni­toring strategies discussed above.

References (1) Colborn, T.; Dumanoski D.; Myers J. P. Our Stolen Fu­

ture. Dutton: New York, NY, 1996. (2) Keith, L. H. Environmental Endocrine Disruptors:A Hand­

book of Property Data, Wiley & Sons: New York, 1997. (3) Hilleman, B. Chem. Eng. News 1998, 76(6), 7-8. (4) Kahn, I. A.; Thomas, P. Neurotoxicology 1997,18, 553-560. (5) Danzo, B. J. Environ. Health Perspect. 1997,105, 294-301. (6) Oosterkamp, A. J.; Hock, B.; Seifert, M.; Irth, H. Trends

Anal. Chem. 1997, 16, 544-553. (7) Safe, S.; Connor, K.; Ramamoorthy, K.; Gaido, K.; Mannes

S. Regul. Toxicol. Pharmacol. 1997, 26, 52-58. (8) vom Saal, F. S.; Timms, G.; Nagel, S.; Dhar, M.; Ganjum,

V; Parmigiani, S.; Weldshons, W. Proc. Natl. Acad. Sci. U.SA. 1997, 94, 2056-2061.

(9) Arnold, S.; Klotz, D.; Collins, B.; Vonier, P.; Guilette, L. Jr.; McLachlan, J. Science 1996, 272, 1489-1492.

(10) Sadik, O. A.; Van Emon, J. M. Biosens. Bioelectron. 1996, 11(8), 1-11.

(11) Sadik, O. A; Van Emon, J. M. CHEMTECH 1997, 27(6), 38-46.

(12) Bender S.; Sadik, O. A. Environ. Sci. Technol. 1998,32(6), 788-797.

(13) Witt, D. M.; Gainer, H. Soc. Neurosci. Abstr. 1998, 639(19), 1632.

(14) Witt, D. M. Regulatory Mechanisms of Oxytocin-Mediated Sociosexual Behavior. In The Integrative Neurobiology of Af­filiation; Carter, C. S., Kirkpatrick, B., Lederhendler, 1.1., Eds.; Annals NY. Acad. Sci. 1997, 807, 287-301.

Omowunmi A. Sadik is an assistant professor in the De­partment of Chemistry, and Diane M. Witt is an assis­tant professor in the Department of Psychology, Behav­ioral Neuroscience Program, at the State University of New York-Binghamton.

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