Environmental endocrine disruptors: Effects on the human male reproductive system.

Incidences of altered development and neoplasia of male reproductive organs have increased during the last 50 years, as shown by epidemiological data. These data are associated with the increased presence of environmental chemicals, specifically "endocrine disruptors," that interfere with normal hormonal action. Much research has gone into testing the effects of specific endocrine disrupting chemicals (EDCs) on the development of male reproductive organs and endocrine-related cancers in both in vitro and in vivo models. Efforts have been made to bridge the accruing laboratory findings with the epidemiological data to draw conclusions regarding the relationship between EDCs, altered development and carcinogenesis. The ability of EDCs to predispose target fetal and adult tissues to neoplastic transformation is best explained under the framework of the tissue organization field theory of carcinogenesis (TOFT), which posits that carcinogenesis is development gone awry. Here, we focus on the available evidence, from both empirical and epidemiological studies, regarding the effects of EDCs on male reproductive development and carcinogenesis of endocrine target tissues. We also critique current research methodology utilized in the investigation of EDCs effects and outline what could possibly be done to address these obstacles moving forward.

Systems biology beyond networks: generating order from disorder through self-organization.

Erwin Schrödinger pointed out in his 1944 book "What is Life" that one defining attribute of biological systems seems to be their tendency to generate order from disorder defying the second law of thermodynamics. Almost parallel to his findings, the science of complex systems was founded based on observations on physical and chemical systems showing that inanimate matter can exhibit complex structures although their interacting parts follow simple rules. This is explained by a process known as self-organization and it is now widely accepted that multi-cellular biological organisms are themselves self-organizing complex systems in which the relations among their parts are dynamic, contextual and interdependent. In order to fully understand such systems, we are required to computationally and mathematically model their interactions as promulgated in systems biology. The preponderance of network models in the practice of systems biology inspired by a reductionist, bottom-up view, seems to neglect, however, the importance of bidirectional interactions across spatial scales and domains. This approach introduces a shortcoming that may hinder research on emergent phenomena such as those of tissue morphogenesis and related diseases, such as cancer. Another hindrance of current modeling attempts is that those systems operate in a parameter space that seems far removed from biological reality. This misperception calls for more tightly coupled mathematical and computational models to biological experiments by creating and designing biological model systems that are accessible to a wide range of experimental manipulations. In this way, a comprehensive understanding of fundamental processes in normal development or of aberrations, like cancer, will be generated.

Fractal analysis in a systems biology approach to cancer.

Cancer is a highly complex disease due to the disruption of tissue architecture. Thus, tissues, and not individual cells, are the proper level of observation for the study of carcinogenesis. This paradigm shift from a reductionist approach to a systems biology approach is long overdue. Indeed, cell phenotypes are emergent modes arising through collective non-linear interactions among different cellular and microenvironmental components, generally described by "phase space diagrams", where stable states (attractors) are embedded into a landscape model. Within this framework, cell states and cell transitions are generally conceived as mainly specified by gene-regulatory networks. However, the system's dynamics is not reducible to the integrated functioning of the genome-proteome network alone; the epithelia-stroma interacting system must be taken into consideration in order to give a more comprehensive picture. Given that cell shape represents the spatial geometric configuration acquired as a result of the integrated set of cellular and environmental cues, we posit that fractal-shape parameters represent "omics" descriptors of the epithelium-stroma system. Within this framework, function appears to follow form, and not the other way around.

Identification of de novo mutations and rare variants in hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is one of the most severe congenital heart malformations, characterized by underdevelopment of the structures in the left heart-aorta complex. The majority of cases are sporadic. Although multiple genetic loci have been tentatively implicated in HLHS, no gene or pathway seems to be specifically associated with the disease. To elucidate the genetic basis of HLHS, we analyzed 53 well-characterized patients with isolated HLHS using an integrated genomic approach that combined DNA sequencing of five candidate genes (NKX2-5, NOTCH1, HAND1, FOXC2 and FOXL1) and genome-wide screening by high-resolution array comparative genomic hybridization. In 30 patients, we identified two novel de novo mutations in NOTCH1, 23 rare patients inherited gene variants in NOTCH1, FOXC2 and FOXL1, and 33 rare patients mostly inherited copy-number variants. Some of the identified variations coexisted in the same patient. The biological significance of such rare variations is unknown, but our findings strengthen the role of NOTCH pathway in cardiac valve development, indicating that HLHS is, at least in part, a 'valve' disease. This is the first report of de novo mutations associated with isolated HLHS. Moreover, the coexistence of multiple rare variants suggests in some cases a cumulative effect, as shown for other complex disease.

On physicalism and downward causation in developmental and cancer biology.

The dominant position in Philosophy of Science contends that downward causation is an illusion. Instead, we argue that downward causation doesn't introduce vicious circles either in physics or in biology. We also question the metaphysical claim that "physical facts fix all the facts." Downward causation does not imply any contradiction if we reject the assumption of the completeness and the causal closure of the physical world that this assertion contains. We provide an argument for rejecting this assumption. Furthermore, this allows us to reconsider the concept of diachronic emergence.

Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration.

We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2'-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix-coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2'-deoxyuridine (dU*)], and the interaction of each hairpin with Mg(2+). All three molecules undergo two-state transitions with melting temperatures (T(M)) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T(M) values of 64-66 degrees C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4-6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36-39 kcal/mol, and favorable entropies of approximately 110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T(M)-dependence on salt yielded slopes of 2.3-2.9 degrees C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na(+) per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg(2+) with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2-4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.

Age-dependent growth inhibition of estrogen-sensitive rat mammary tumors. Probable role of alpha-fetoprotein.

The growth patterns of estrogen (E)-sensitive mammary tumors (SNMU) and autonomous mammary tumors (ANMU) were compared in normal host F344 and BUF rats of different ages. Both SNMU and ANMU tumors have comparable amounts of E receptors of similar physicochemical properties. The E sensitivity was apparent in SNMU tumors regardless of the age of inoculation. The latency period before tumors became palpable was considerably lengthened when the SNMU tumors were inoculated into newborn hosts. The growth pattern of the ANMU tumor was not affected by the age or sex of the host. Comparable results previously obtained with two hypophyseal cell lines suggest that this growth pattern could now be extended to other E-sensitive autonomous systems and become a generalized principle. These results suggest that: 1) the presence of 17 beta-estradiol (E2) receptors is not sufficient to qualify cells to be considered E-sensitive, 2) E-sensitive cells may be delayed in expressing their malignant growth properties if inoculated during the perinatal period, and 3) alpha-fetoprotein may be the substance responsible for the inhibition of the growth of E-sensitive tumor cells during the perinatal stage.

Growth inhibition of estrogen-sensitive rat mammary tumors. Effect of an alpha-fetoprotein-secreting hepatoma.

The growth and regression patterns of estrogen (E)-sensitive mammary tumors (SNMU) and autonomous mammary tumors (ANMU) were studied in normal, castrated, and alpha-fetoprotein (AFP)-secreting and nonsecreting hepatoma-bearing female BUF rats. Both SNMU and ANMU tumors had comparable amounts of E receptor. SNMU tumors grew faster in female hosts than in male hosts. The latency period of SNMU tumors was lengthened when the tumor was inoculated into newborn hosts. These E-sensitive tumors regressed after castration of the adult hosts. Tumor regression was also observed in SNMU-bearing rats inoculated with AFP-secreting hepatomas. The growth pattern of the ANMU tumor was not affected by the sex or age of the host. Castration and inoculation of hepatomas to ANMU tumor-bearing rats did not result in regression of the ANMU tumors. We suggest that AFP inhibited the growth of E-sensitive cells in newborn and hepatoma 7777-bearing hosts. This inhibitory effect of AFP could not be attributed to a "functional castration" resulting from the trapping of 17 beta-estradiol (E2) by AFP, because the plasma levels of E2 in these animals affect levels of gonadotropins. Moreover, castration of hepatoma 7777-bearing rats resulted in increased plasma follicle-stimulating hormone and luteinizing hormones levels. The results strongly suggested that a) the malignant properties of E2-sensitive cells in adult hosts can be controlled by AFP, and b) the mechanism by which AFP inhibits the growth of E2-sensitive cells is independent of the E2 "trapping" phenomenon. An ontogenetic approach to the mechanism of action of estrogens may account for available results.

Estrogen-sensitive proliferation pattern of cloned Syrian hamster kidney tumor cells.

Estrogen-sensitive hamster kidney tumor cells H301 and their clonal derivatives were inhibited from proliferating in culture by charcoal-dextran (CD) stripped serum in a dose-dependent manner. Homologous serum was more potent an inhibitor than heterologous (human, bovine, equine) sera. Natural and synthetic estrogens failed to increase the proliferation rate of cells maintained in 2% CD Syrian hamster serum (SHS). At CDSHS concentrations above 2%, cell proliferation was significantly inhibited and estrogens completely reversed this inhibitory effect. Nonestrogenic steroids failed to overcome the serum inhibition. Two synthetic estrogens, moxestrol and 11 beta-chloromethylestradiol, were 10-fold more potent than estradiol in increasing cell proliferation yields; they were however, less potent than estradiol in inhibiting [3H]estradiol binding to intracellular estrophilins. d-Equilenin, a poor inducer of kidney tumors, was a weak estrogen in the "in culture" proliferation assay. Ethynylestradiol was highly estrogenic in culture while reports suggest that it is poor tumor inducer in the animal. Progestagens inhibit the growth of estrogen-induced kidney tumors; only promegestone partially blocked the proliferative effect of estradiol in cultures supplemented with 10% CDSHS. Charcoal-dextran stripped serum from animals bearing a diethylstillbestrol implant was as effective as the serum of untreated male hamsters in inhibiting the proliferation of B3H301 cells. These results are compatible with the following interpretations: (a) hamster serum contains a potent specific inhibitor of the proliferation of estrogen-sensitive cells (estrocolyone); (b) estrogens induce cell proliferation by neutralizing the effect of this serum-borne inhibitor; (c) the poor correlation between estrophilin binding and proliferative potency suggests no direct estrophilin involvement in the proliferative effect of estrogens on these cells; (d) the results obtained in this "in culture" model using estrogen (except ethynylestradiol) and other steroids are compatible with the results obtained in the animal; and (e) the tumorigenic process in Syrian hamster kidneys triggered by estrogens probably involves their direct interaction within these cells (shut-off effect) in addition to the neutralization of the estrocolyone.

Negative controls of cell proliferation: human prostate cancer cells and androgens.

LNCaP cells represent a useful tool to explore the mechanism of sex hormone action on cell proliferation in an "in culture-in animal" model. Results indicated that: (a) these cells were inhibited from proliferating for extended periods (up to 30 days) when placed in charcoal-dextran-stripped sera; they remained, however, viable because they proliferated when sex hormones were added to this medium; (b) the inhibitory effect of sera was reversed by the addition of 5 alpha-dihydrotestosterone at 3 x 10(-10) M, 17 beta-estradiol at 3 x 10(-8) M and higher concentrations, and progesterone at 3 x 10(-10) M and higher concentrations; (c) while the dose response to androgens was biphasic (i.e., 5 alpha-dihydrotestosterone at concentrations higher than 3 x 10(-10) M resulted in progressively lower cell yields), estrogens and progestagens exhibited a monophasic pattern; (d) these cells were exceedingly sensitive to the nutritional environment in which they grew; (e) while these cells have androgen receptors (68 fmol/mg protein; Kd = 2 x 10(-9) M), estrogen and progestagen receptors could not be detected by biochemical and immunocytochemical techniques; (f) tumors grew at the site of inoculation in castrated nude mice carrying 17 beta-estradiol and progesterone pellets and in intact male nude mice implanted with placebo pellets, while tumors did not grow in castrated nude mice implanted with a 5 alpha-dihydrotestosterone pellet. Taken together the data collected are compatible with the following conclusions: (a) the proliferative response in LNCaP cells seems not to be directly mediated by their intracellular androgen receptors; (b) plasma-borne trypsin-sensitive inhibitors of the proliferation of these cells (androcolyone I) appear to play a significant role in the proliferative event; (c) natural and synthetic androgens, estrogens, and progestagens cancelled the inhibition by charcoal-dextran-stripped human sera; (d) only androgens were able to trigger an inhibition of cell proliferation (shutoff effect) at concentrations higher than those that affected maximal cell yields (direct negative hypothesis); and (e) a faulty shutoff response is probably a crucial event for the tumorigenesis of these human prostate cells.

Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.

The human breast tumor cloned cell lines T47D-A8 and All are estrogen dependent for cell proliferation in the nude mouse model. In contrast, these cells multiplied at similar rates when grown in serum-free cultures, regardless of the presence of 17 beta-estradiol (3 X 10(-11) to 3 X 10(-8) M estradiol). Addition of 10% charcoal-dextran stripped human female serum to the culture medium resulted in a marked inhibition of cell proliferation. The addition of 3 X 10(-11) M estradiol overcame the inhibitory effect of serum. Similar results were obtained with the human breast tumor C7MCF7 cell line. Both cell lines contain similar estrophilin levels. The Kd of the estrophilin-estradiol complex was 0.39 X 10(-10) M for C7MCF7 cells and 4.4 X 10(-10) M for T47D-A11 cells. Maximal cell yields were achieved at 5 X 10(-12) M free estradiol levels in 10% charcoal-dextran stripped serum supplemented medium. These data are compatible with the following interpretation: (a) estradiol-sensitive cells are inhibited from proliferating by a serum-borne factor; and (b) estradiol neutralizes this inhibitory effect. This mechanism seems not to be mediated by estradiol binding to the cellular estrophilins because (a) the free estradiol levels needed for maximal response are significantly lower than the estrophilin Kds, and (b) maximal proliferation rates occur at similar estradiol concentrations for these three cell lines, regardless of the binding properties of their estrophilins.

Estrogen-induced cell multiplication: direct or indirect effect on rat uterine cells?

It has been previously demonstrated that the exposure of the liver to estradiol-17 beta (E2) is sufficient to produce cell multiplication in the oviduct of the quail. In this report we determine whether this can also be observed in the rat. Ovariectomized, primed, adult rats were infused over a 3-h period with 2 micrograms E2/kg BW via the jugular vein (systemically). Mitotic figures arrested by colchicine appeared in the uterine luminal epithelium 16-20 h later and remained at maximal levels for the subsequent 3 intervals of 4 h. The minimum dose required to induce a mitotic response was 1.25 micrograms E2/kg BW. There was no increase in response with larger doses. Plasma E2 levels increased linearly with the dose infused systemically. Under the same conditions, E2 infused via the spleen produced mitoses with a similar time course and dose response. However, the amplitude of the response and the plasma E2 levels were significantly depressed. To circumvent problems involved with the interpretation of the results obtained by splenic infusions, we infused 2 micrograms E2/kg BW via the superior mesenteric vein (hepatoportal system). There was no mitotic response and the plasma E2 levels were not elevated above controls. We conclude that a strictly liver mediated, indirect mechanism for the regulation of cell multiplication proposed for quail, is not operative in the rat. In an initial attempt to explain this difference between species, we injected 5 micrograms E2/kg BW sc into hypophysectomized rats. Hypophysectomy had no effect upon the proliferation of the uterine luminal epithelial cells.

The proliferative effect of "anti-androgens" on the androgen-sensitive human prostate tumor cell line LNCaP.

The effect of steroidal and nonsteroidal "anti-androgens" on the proliferative capacity of androgen-sensitive LNCaP-FGC human prostate tumor cells in culture was studied using charcoal-dextran stripped human serum-supplemented media. Cyproterone and medroxyprogesterone acetates, flutamide, hydroxyflutamide, and anandron (R23908) were administered alone at concentrations between 3 X 10(-12) and 3 X 10(-6) M. Results indicated that although medroxyprogesterone induced maximal proliferation at 3 X 10(-9) M, the other "anti-androgens" (with the exception of flutamide that was ineffective) were effective at 3 X 10(-8) M and higher concentrations; the amplitude of the proliferative response by these compounds was comparable to that elicited by estradiol-17 beta (3 to 5-fold over control). None of the anti-androgens tested triggered the shutoff effect characteristic of androgen action. When 3 X 10(-10) M DHT and the above mentioned anti-androgens were administered simultaneously, a synergistic pattern was seen; on the contrary, 3 X 10(-8) M DHT cancelled the proliferative effect of each of the anti-androgens when administered simultaneously. The relative binding affinity of these anti-androgens to androgen receptors present in LNCaP-FGC cells did not correlate well with their proliferative efficiency. The data collected were interpreted within the premises of the negative control hypotheses for the regulation of cell proliferation in metazoans. Within those premises, results became compatible with the notion that first, "anti-androgens" elicited the proliferation of androgen-sensitive cells by neutralizing the effect of a serum-borne inhibitor (androcolyone-I); this event seems not to be mediated by androgens receptors. Second, anti-androgens did not trigger a proliferative shutoff response like androgens do, i.e. the proliferative pattern induced by anti-androgens was comparable to that elicited by estrogens and progestins. Third, when administered simultaneously with 3 X 10(-10) M DHT, anti-androgens behaved synergistically. Fourth, the DHT-induced shutoff effect consistently overrode the proliferative effect generated by anti-androgens and estrogens when added alone. Finally, taken together these results raise important questions regarding the therapeutic role of anti-androgens in prostate cancer.

Biological effects of sex hormone-binding globulin on androgen-induced proliferation and androgen metabolism in LNCaP prostate cells.

The effects of purified human sex hormone-binding globulin (SHBG) on androgen-sensitive cell proliferation were examined using a human prostatic cell line (LNCaP-FGC). Cells were grown for 5 days in medium supplemented with 10% charcoal-dextran-stripped human serum (10% CDHuS) and various concentrations of 5 alpha-dihydrotestosterone (DHT). In 10% CDHuS, without SHBG, the proliferative response of these cells to androgens was typically biphasic. At low androgen concentrations, cell yields were increased in a dose-dependent manner, reaching maximal levels at 0.3 nM DHT. However, at high androgen concentrations, cell proliferation was inhibited. Addition of purified human SHBG to the medium reduced the effectiveness of DHT on both phases of the proliferative response in a dose-dependent manner. These effects of SHBG appeared to be due primarily to the high affinity binding of DHT by SHBG. Proliferative responses induced by the synthetic androgen methyltrienolone (R1881), which binds poorly to SHBG, were not affected by added SHBG. Furthermore, analysis of the protein binding of DHT revealed that cell proliferation correlated best with the concentration of DHT not bound to SHBG. The presence of SHBG in the medium also altered the uptake and metabolism of DHT. LNCaP-FGC cells rapidly metabolized DHT to a polar glucuronidase-sensitive conjugate of DHT. In 10% CDHuS, LNCaP-FGC cells conjugated virtually all of the added DHT during the 5-day experiment. However, in medium containing SHBG, the SHBG-bound DHT remained unconjugated; more than 90% of the DHT initially bound to SHBG was present in the medium at the end of the experiment as unconjugated DHT. Uptake of radiolabeled DHT by cells was also inhibited by SHBG. In summary, these experiments provide evidence that 1) SHBG-bound DHT is not a signal for DHT-induced cell proliferation and 2) SHBG inhibits the uptake and metabolism of DHT by LNCaP-FGC cells.

Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells transfected with androgen receptor.

Sex steroids control the proliferation of their target cells through two different pathways: 1) proliferative response (Step-1); and 2) inhibition of cell proliferation (Step-2). Mechanisms of cell proliferation regulation are incompletely understood; however, there is general agreement with the notion that sex steroid receptors play an important role in the control of the proliferation of sex steroid target cells. To test this hypothesis, a full human androgen receptor (AR) vector was transfected into human breast cancer MCF7 cells. The cloned cells that stably express the AR, called MCF7-AR1 cells, contained approximately five times more AR than the wild-type MCF7 cells from which they were derived. These AR-transfected cells retained their capacity to proliferate when estrogens were added to 10% charcoal-dextran stripped human serum but did not acquire the ability to proliferate when androgens were added to this medium. In serumless medium (ITDME), these cells proliferated maximally, as MCF7 cells did; however, natural and synthetic androgens prevented the AR-transfected cells from proliferating. Inhibition of cell proliferation occurred when physiological androgen concentrations (1 nM) were added to ITDME; this effect was almost completely reversed by Casodex, a synthetic androgen antagonist. Under the effect of androgens added to ITDME, MCF7-AR1 cells were arrested in the G0/G1 phase within 24 h. These data suggest that: 1) the androgen-induced inhibition of cell proliferation (Step-2) is AR-mediated; and 2) the AR may be necessary, but not sufficient, to mediate the androgen-induced proliferative response (Step-1).

Human serum does not contain a high affinity estrogen-binding glycoprotein different from sex hormone-binding globulin.

The mannoglycoprotein fraction obtained by Concanavalin-A chromatography of human serum binds both androgens and estrogens with high affinity. Sex hormone-binding globulin (SHBG) is a component of this fraction that binds both steroids, but the fraction may contain another component that binds only estrogen. We used several chromatographic methods to ascertain whether the estradiol-binding properties of the mannoglycoprotein fraction could be attributed to SHBG or to SHBG and the putative estrogen-binding protein. DEAE-trisacryl, chromatofocusing, and anti-SHBG-immunoglobulin Sepharose chromatography resulted in coelution of the androgen- and estrogen-binding activities. SHBG purified by ligand affinity chromatography as well as immunoaffinity-purified SHBG had estradiol-binding properties similar to those of the crude mannoglycoprotein preparation. These data strongly suggest that 1) SHBG is the only estradiol-binding protein in the mannoglycoprotein fraction obtained by Concanavalin-A chromatography of human serum, and 2) the putative estradiol-binding protein of serum is most likely SHBG.

Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSY NMR experiment.

The binding of ethidium bromide to a DNA hairpin (dU(5)-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1. 60x10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin, consistent with complex formation (1.22x10(-6) cm(2)/s at 26 degrees C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU(5)-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution.

The pesticides endosulfan, toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitive cells.

Estrogenic pesticides such as DDT and chlordecone generate deleterious reproductive effects. An "in culture" bioassay was used to assess the estrogenicity of several pesticides. The E-screen test uses human breast estrogen-sensitive MCF7 cells and compares the cell yield achieved after 6 days of culture in medium supplemented with 5% charcoal-dextran stripped human serum in the presence (positive control) or absence (negative control) of estradiol and with diverse concentrations of xenobiotics suspected of being estrogenic. Among the organochlorine pesticides tested, toxaphene, dieldrin, and endosulfan had estrogenic properties comparable to those of DDT and chlordecone; the latter are known to be estrogenic in rodent models. The E-screen test also revealed that estrogenic chemicals may act cumulatively; when mixed together they induce estrogenic responses at concentrations lower than those required when each compound is administered alone.

p-Nonyl-phenol: an estrogenic xenobiotic released from "modified" polystyrene.

Alkylphenols are widely used as plastic additives and surfactants. We report the identification of an alkylphenol, nonylphenol, as an estrogenic substance released from plastic centrifuge tubes. This compound was extracted with methanol, purified by flash chromatography and reverse-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Nonylphenol induced both cell proliferation and progesterone receptor in human estrogen-sensitive MCF7 breast tumor cells. Nonylphenol also triggered mitotic activity in rat endometrium; this result confirms the reliability of the MCF7 cell proliferation bioassay. The estrogenic properties of alkylphenols, specifically nonylphenols, indicate that the use of plasticware containing these chemicals in experimental and diagnostic tests may lead to spurious results, and these compounds as well as alkylphenol polyethoxylates may also be potentially harmful to exposed humans and the environment at large.

Developmental effects of endocrine-disrupting chemicals in wildlife and humans.

Large numbers and large quantities of endocrine-disrupting chemicals have been released into the environment since World War II. Many of these chemicals can disturb development of the endocrine system and of the organs that respond to endocrine signals in organisms indirectly exposed during prenatal and/or early postnatal life; effects of exposure during development are permanent and irreversible. The risk to the developing organism can also stem from direct exposure of the offspring after birth or hatching. In addition, transgenerational exposure can result from the exposure of the mother to a chemical at any time throughout her life before producing offspring due to persistence of endocrine-disrupting chemicals in body fat, which is mobilized during egg laying or pregnancy and lactation. Mechanisms underlying the disruption of the development of vital systems, such as the endocrine, reproductive, and immune systems, are discussed with reference to wildlife, laboratory animals, and humans.