Familiar and novel reproductive endocrine disruptors: xenoestrogens, dioxins and nanoparticles.

Environmental contaminants are known to exert endocrine-disrupting effects on the reproductive axis of animals. Many of these molecules can affect steroid biosynthesis or estrogen-receptor signaling by behaving as estrogen-like molecules ("xenoestrogens"), or by exerting estrogenmodulatory effects. Exposure to some compounds has been correlated with the skewing of sex ratios in aquatic species, feminization and demasculinization of male animals, declines in human sperm counts, and overall diminution in fertility of birds, fish, and mammals. We herein devote space to several classes of endocrine-disrupting compounds (EDCs), including estrogenic substances such as bisphenol A (BPA), molecules that can behave at times anti-estrogenically while activating the aromatic hydrocarbon receptor (AHR), such as dioxins (a known human carcinogen), and novel, ubiquitous molecules such as nanoparticles, particularly gold nanoparticles (GNPs), that appear to alter the sexsteroid biosynthetic pathway.

Teratogenic effects of ethanol exposure on zebrafish visual system development.

Ethanol intake during pregnancy can produce a wide range of adverse effects on nervous system development including fetal alcohol syndrome (FAS). The most severe congenital malformation observed in newborns with FAS is cyclopia. In this study, we have exposed zebrafish embryos to different ethanol concentrations (2.4%, 1.5% or 1.0%) during eye morphogenesis in four zebrafish strains (AB, EK, GL and TL). In addition, we have studied the survival rate of the cyclopic animals to the end of larval development. The zebrafish strains GL and AB generated the higher percentage of cyclopic animals after exposure to 2.4% ethanol, while EK showed the higher percent cyclopic animals using 1.5% and 1.0% ethanol. The EK strain showed the higher percent survival during the larval period at all ethanol concentrations (2.4%, 1.5% and 1.0%). Moreover, we have investigated cytoarchitectural alterations in the main components of the visual pathway-retina and optic tectum-and ethanol treatment affects both the retina and the optic tectum. The lamination of neural retina is clearly delayed in treated larvae 3 days postfertilization and the thickness of the pigmented epithelium is considerably reduced. With regard to the optic tectum, treatment with ethanol alters the normal pattern of tectal lamination. The use of zebrafish EK strain is a suitable in vivo vertebrate model system for analyzing the teratogenic effect of ethanol during vertebrate visual system morphogenesis as it relates to both cyclopia and FAS.

Environmental toxicants and effects on female reproductive function.

One of the most toxic substances known to humans, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), is also highly pervasive in the environment. It is created naturally in volcanic eruptions and forest fires, and anthropogenically in waste incineration, chlorination processes and certain plastics manufacture. From reports of large industrial and other accidents, or from experimental studies, dioxin exposure has been correlated in animal models and/or humans with chloracne of the skin, organ cancers, hepatotoxicity, gonadal and immune changes, pulmonary and other diseases such as diabetes, skewing of the sex ratio, and infertility. We have demonstrated that the aromatic hydrocarbon receptor (AHR) that binds dioxin in tissues is localized in zebrafish, rat and rhesus monkey (Macaca mulatta) ovaries and in rat and human luteinizing granulosa cells (GC) (among other tissues), that labeled dioxin is specifically localized to granulosa cells of the ovarian follicle as observed by autoradiography, and that incubations of GC or ovarian fragments with environmentally relevant concentrations (fM to nM) of dioxin inhibit estradiol secretion significantly. Our experiments show that in human, non-human primate, rat, trout, and zebrafish ovarian tissues, dioxin inhibits estrogen synthesis at some level of the steroid biosynthetic pathway, most likely by inhibiting transcription of mRNAs for or activity of side-chain cleavage (Cyp11a1 gene) and/or aromatase (Cyp19a1 gene) enzymes, or conceivably other steroidogenic enzymes/factors. Such an untoward effect on estrogen synthesis in females exposed to dioxin environmentally may predispose them to defects in aspects of their fertility.

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio).

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.

Oxidative stress in zebrafish cells: potential utility of transgenic zebrafish as a deployable sentinel for site hazard ranking.

In order to quickly assess potential environmental hazards of forwardly deployed military bases, we have focussed our efforts on biochemical and molecular changes in vertebrate cells following exposure to aqueous soil extracts. To this end, we are designing a series of deployable transgenic fish. Fish exhibit many of the same general defenses against toxic chemicals as do mammals, including enzyme induction, and the generation of oxidative stress. In response to many foreign compounds that generate oxidative stress, the transcription of certain protective genes is induced via specific DNA motifs called electrophile response elements (EPREs). We have made a plasmid construct containing a single murine EPRE fused to a minimal promoter and the cDNA encoding firefly luciferase (EPRE-LUC). In this paper, we have shown that the treatment of zebrafish cell line ZEM2S with a variety of chemicals known to induce EPRE-dependent transcription in cultured mammalian cells, results in dose-dependent induction of the transiently-transfected EPRE-LUC reporter construct. Compounds tested include aromatic hydrocarbons, heavy metals, and organophosphates. We observed similar dose-dependent responses when we treated ZEM2S and human cells in vitro with identical aqueous extracts of soil from hazardous waste sites. This suggests that the mechanism by which these compounds activate transcription is well conserved between mammals and zebrafish, and that transgenic zebrafish lines containing EPRE-driven reporter constructs might be useful as sentinels for the early detection of oxidative stress-inducing chemicals.

Transgenic zebrafish as sentinels for aquatic pollution.

Using the golden mutant zebrafish having a decrease in interfering pigmentation, we are developing transgenic lines in which DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed. We have begun with three response elements that recognize three important classes of foreign chemicals. Aromatic hydrocarbon response elements (AHREs) respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. Soon, we will include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the effects of retinoids in the environment. Each of these substances is known to be bioconcentrated in fish to varying degrees; for example, 10(-17) M TCDD in a body of water becomes concentrated to approximately 10(-12) M TCDD in a fish, where it would act upon the AHRE motif and turn on the luciferase (LUC) reporter gene. The living fish as a sentinel will not only be assayed intact in the luminometer, but--upon several days or weeks of depuration--would be usable again. To date, we have established that zebrafish transcription factors are able to recognize both mammalian and trout AHRE, EPRE, and MRE sequences in a dose-dependent and chemical-class-specific manner, and that expression of both the LUC and jellyfish green fluorescent protein (GFP) reporter genes is easily detected in zebrafish cell cultures and in the intact live zebrafish. Variations in sensitivity of this model system can be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core consensus response element and flanking regions. This transgenic technology should allow for a simple, exquisitely sensitive, and inexpensive assay for monitoring aquatic pollution. We have already initiated studies using sentinel zebrafish to monitor a public drinking water source.

"Gene-swap knock-in" cassette in mice to study allelic differences in human genes.

Genetic differences in environmental toxicity and cancer susceptibility among individuals in a human population often reflect polymorphisms in the genes encoding drug-metabolizing enzymes (DMEs), drug transporters, and receptors that control DME levels. This field of study is called "ecogenetics", and a subset of this field--concerning genetic variability in response to drugs--is termed "pharmacogenetics". Although human-mouse differences might be 3- to perhaps 10-fold, human interindividual differences can be as great as 20-fold or more than 40-fold. It would be helpful, therefore, to study toxicokinetics/pharmacokinetics of particular environmental agents and drugs in mice containing these "high-" and "low-extreme" human alleles. We hope to use transgenic "knock-in" technology in order to insert human alleles in place of the orthologous mouse gene. However, the knock-in of each gene has normally been a separate event requiring the following: (a) construction of the targeting vector, (b) transfection into embryonic stem (ES) cells, (c) generation of a targeted mouse having germline transmission of the construct, and (d) backcross breeding of the knock-in mouse (at least 6-8 times) to produce a suitable genetically homogeneous background (i.e., to decrease "experimental noise"). These experiments require 1 1/2 to 2 years to complete, making this very powerful technology inefficient for routine applications. If, on the other hand, the initial knock-in targeting vector might include sequences that would allow the knocked-in gene to be exchanged (quickly and repeatedly) for one new allele after another, then testing distinctly different human polymorphic alleles in transgenic mice could be accomplished in a few months instead of several years. This "gene-swapping" technique will soon be done by zygotic injection of a "human allele cassette" into the sperm or fertilized ovum of the parental knock-in mouse inbred strain or by the cloning of whole mice from cumulus ovaricus cells or tail-snip fibroblasts containing the nucleus wherein each new human allele has already been "swapped." In mouse cells in culture using heterotypic lox sites, we and others have already succeeded in gene swapping, by exchanging one gene, including its regulatory regions, with a second gene (including its regulatory regions). It is anticipated that mouse lines carrying numerous human alleles will become commonplace early in the next millennium.

Characterization of the MTF-1 transcription factor from zebrafish and trout cells.

The metal response element (MRE)-binding transcription factor-1, MTF-1, is a zinc-responsive protein that controls transcription of metallothionein (MT) genes in many cell types. In addition, MTF-1 is also hypothesized to regulate transcription of a battery of genes involved in the defense against oxidative stress. Manipulating the Zn concentration in the low microM range reversibly modulates the DNA-binding activity of the mammalian MTF-1; this effect is inhibited at low temperature. This report examines the presence and binding properties of MTF-1 in cell lines derived from warm- and cold-water fishes (zebrafish and trout, respectively). We found that both species of fish express MRE-specific binding activities that are immunologically similar to mouse MTF-1. MTF-1-binding from the cells of both species of fish was activated when cells were treated with Zn but not with Cd. Zebrafish cells contained a single isoform of MTF-1 with binding properties similar to mammalian MTF-1. Trout cells, on the other hand, contained two isoforms of MTF-1: MTF-1H and MTF-1L. Zn reversibly modulated MTF-1H binding in a temperature-dependent manner. Similarly, Zn reversibly modulated MTF-1L binding, but, in contrast, such modulation occurred readily at 4 degrees C. This data demonstrate the conservation of binding specificity, binding properties, and regulation of MTF-1 in fishes.

Activation of transcription factors in zebrafish cell cultures by environmental pollutants.

Many classes of environmental pollutants are found at significant levels in the aquatic environment. We are designing a fish model as an inexpensive and efficient system for the assessment of aquatic pollution. Three classes of environmental pollutants-halogenated and nonhalogenated aromatic hydrocarbons, heavy metals, and potent electrophiles-are known to upregulate particular mammalian genes via the activation of specific DNA motifs called aromatic hydrocarbon (AHREs), heavy metal (MREs), and electrophile (EPREs) response elements, respectively. We have made plasmid constructs, using these mammalian or trout response elements to drive the luciferase reporter gene. Here we show that transient transfection of the zebrafish ZEM2S cell line with these reporter constructs imparts dose-dependent gene induction upon exposure to a variety of chemicals within each of these three classes of inducers: [a] (AHRE-mediated) 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, 3,4,5,3',4',5'-hexabromobiphenyl, Aroclor 1254, and benzo[a]pyrene; [b] (MRE-mediated) Cd(2+), Zn(2+), Hg(2+), and Al(3+); and [c] (EPRE-mediated) tert-butylhydroquinone, Hg(2+), Pb(2+), As(3+), Cu(2+), and Cd(2+). As expected, some agents gave a response to only one of the three classes, whereas others gave a mixed (AHRE- plus EPRE-mediated or MRE- plus EPRE-mediated) response. In response to several environmental agents, we found that differences in the electrophoretic mobility shift assay, using the AHRE or MRE as probe, were consistent with the degree of transcriptional activation seen with the reporter constructs. Our data suggest that these reporter constructs might be valuable for the generation of transgenic zebrafish in order to carry out mechanistic and developmental studies of transcriptional activation by environmental contaminants; moreover, such transgenic zebrafish lines might be useful as a sentinel for assessing aquatic pollution.

Dose-dependent induction of the microsomal monooxygenase system by phenobarbital and 3-methylcholanthrene in the ad libitum and calorie-restricted female rat.

1. We characterized the dose-dependent induction of the microsomal monooxygenase system by phenobarbital (PB) and 3-methylcholanthrene (MC) in the female Fischer 344 rat, which was either calorie restricted (CR) or fed ad libitum (AL). 2. Maximal induction of the major inducible isozymes (2B1/2B2 or 1A1) in rat was achieved at the lowest of the inducer doses employed (10 mg/kg body weight) in both feeding groups. 3. The patterns of dose-dependent PB induction and its magnitude differed between total P450 induction and induction of catalytic activities in AL and CR groups, whereas no differences between CR and AL rat were found in either spectrally detected P450 or EROD activity patterns of dose-dependent MC induction. 4. Calorie restriction increased the inducibility of some hepatic drug-metabolizing enzyme activities. 5. Monoclonal antibody-directed inhibition of MC-induced ethoxyresorufin O-deethylation (EROD) was 55-60% at all induction levels in AL rat and 65-70% in CR rat, while MAb inhibition of PB-induced pentoxyresorufin O-depenthylation (PROD) averaged about 55% in AL and 60% in CR rat.

Effects of benzo(a)pyrene and tetrachlorodibenzo(p)dioxin on fetal dolphin kidney cells: inhibition of proliferation and initiation of DNA damage.

Dolphin kidney cells (CDK) were exposed in vitro to benzo(a)pyrene (BaP) in the presence or absence of 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), a cytochrome P450-inducing agent, and/or alpha-naphthoflavone (alpha NF), an inhibitor of cytochrome P450 induction. BaP inhibited mitosis in CDK cells in a dose-dependent manner. TCDD, while inhibiting cell proliferation, did not show a strict dose-dependent mode of action. BaP inhibition of mitosis was decreased by alpha NF, which also decreased the inhibitory effects of TCDD on CDK proliferation. BaP treatment initiated both 3H-thymidine incorporation and the increased alkali lability of DNA functions of the initiation of excision repair. Cells pre-treated with TCDD and then exposed to BaP exhibited increased BaP-DNA adduct levels and increased DNA excision repair. These data indicate that dolphin cells metabolized BaP in vitro as a function of cytochrome P450-associated activities, that BaP metabolites covalently bound to cellular DNA and initiated excision repair. Inhibition of the cytochrome P450-mediated metabolism of BaP decreased the BaP-associated inhibition of mitosis in dolphin cells.

Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet.

Ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylase activities, and contributions of the P450 cytochromes CYP1A1, CYP2B1 and 2, CYP2C6 and CYP2C12 to these metabolic activities, were evaluated in hepatic microsomes from ad libitum and calorie restricted female Fischer 344 rats across an age continuum from 1 to 26 months. The presence of CYP1A in microsome preparations was confirmed by western blot analysis. Uninduced levels of EROD and PROD peak in very young animals, decline to about 3 months of age, and do not exhibit an additional substantive decline after about 3 months of age. Monoclonal antibody (MAb) 1-7-1 (anti-CYP1A) strongly inhibited EROD activity in all microsome preparations, with the highest levels of inhibition in microsomes from 1- and 3-month-old AL animals. PROD activity in 1-month uninduced animals was apparently not attributable solely to CYP2B1 and 2, since it was inhibited by about 30% in both 1- and 26-month-old AL rats by an MAb specific for CYP2C12. However, in CR rats, CYP2C12 did not contribute to PROD activity. Approximately 40% of PROD activity in old AL rats and 20% of PROD activity in old CR rats was inhibited by an MAb specific for CYP2C6. These data indicate that long-term calorie restriction modulates either the expression or post-translational modification patterns of constitutive P450 isozymes in rats as they age, with P450 patterns in calorie restricted rats more closely resembling those found in young animals.