Effects of endocrine disrupting compounds on the pathology and oestrogen receptor alpha and beta distribution in the uterus and cervix of ewe lambs.

A number of chemicals have been classed as endocrine disrupting compounds due to their ability to mimic the actions of endogenous hormones in vivo and in vitro. The objective of this experiment was to determine the pathological changes and oestrogen receptor (ER) distribution in the cervix and uterus of prepubertal ovariectomised ewe lambs following exposure to a range of compounds with a predominantly oestrogenic effect. Lambs were exposed to diethylstilbestrol (0.175 mg/kg biweekly), bisphenol-A (3.5mg/kg biweekly) or octylphenol (3.5mg/kg biweekly) for 6 weeks. Following sacrifice, uterine and cervical tissue pathology was assessed. The endometrial and myometrial areas were quantified and the distribution of ERalpha and ERbeta assessed by immunohistochemistry. No differences were observed between control and octylphenol-exposed lambs in uterine gross pathology and histopathology. Uteri from bisphenol-A- and diethylstilbestrol-exposed lambs were heavier than both control and octylphenol-exposed lambs. In the bisphenol-A-exposed lambs, endometrial oedema accounted for a significant increase in the endometrial cross-sectional area over the other groups. Uteri from animals exposed to diethylstilbestrol showed variable pathology including oedema and cellular proliferation. Keratinisation of the cervical epithelium was observed in both bisphenol-A- and diethylstilbestrol-exposed lambs. Exposure to diethylstilbestrol and bisphenol-A was associated with a diffuse intracellular distribution of ERalpha and ERbeta in the uterine endometrium. This was in addition to the strong cytoplasmic staining of uterine epithelial cells and nuclear staining of specific sub-epithelial cells observed in all groups. We conclude that a 6-week exposure of lambs to bisphenol-A and diethylstilbestrol altered the uterocervical environment and has the potential to disrupt subsequent reproductive function. Pathological changes could not be detected in the uterus or cervix of lambs exposed to octylphenol.

Application of genomics to the definition of the molecular basis for toxicity.

Transcript profiling technology enables quantitative measurement of the transcriptional activity of potentially thousands of genes in biological samples. The application of such technology to toxicology, toxicogenomics, promises substantial dividends in mechanistic toxicity research and also, possibly, the ability to predict adverse toxicity for novel or untested compounds. Our laboratory has developed a custom approach to this technology, designing cDNA microarray platforms specifically for gene expression events of relevance to a large number of toxicological endpoints. Such arrays allow comprehensive coverage of genes associated with entire pathways (such as oxidative stress, signal transduction, stress response, epithelial biology) and enable simultaneous measurement of more than ten thousand gene expression events.

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.

Differential activation by xenoestrogens of ER alpha and ER beta when linked to different response elements.

The discovery of a second estrogen receptor (ER beta) has significant implications for our understanding of the molecular basis for the diverse actions of estrogen. Here we report the differential activation by natural and xenobiotic estrogens of ER alpha and ER beta when linked to different response elements. Receptor mediated activation of reporter constructs containing either the estrogen response element (ERE) from the vitellogenin (Vit) gene or from the luteinizing hormone beta (LH) gene were examined in transiently transfected Cos-1 cells. ER beta preferentially activated the consensus Vit ERE whereas ER alpha showed greater activation at the divergent LH ERE. This differential activation was observed for a number of ligands including estradiol, estrone, bisphenol A, octylphenol and diethystilbestrol. These findings show that the nature of the ERE, as well as the ratio of ER subtypes in a particular cell/tissue, will influence whether particular estrogen responsive genes are activated in the presence of natural or xenobiotic estrogens.

Amino acid residues in both the DNA-binding and ligand-binding domains influence transcriptional activity of the human peroxisome proliferator-activated receptor alpha.

We have investigated the basis of the lack of activity of a natural variant human peroxisome proliferator-activated receptor alpha, hPPARalpha6/29. A subcloning approach was used to change the four variant amino acids in the hPPARalpha6/29 sequence, individually and in combination, to those found in an active human PPARalpha. Individual amino acid "back mutations" were unable to confer on hPPARalpha6/29 the ability to be activated by peroxisome proliferators in a transient transfection assay. Although hPPARalpha6/29 was able to bind specifically to DNA in the presence of the retinoid X receptor alpha (RXRalpha), the complete restoration of receptor transcriptional activity required two separate back mutations of the hPPARalpha6/29 sequence, namely amino acid 123 in the DNA binding domain, and amino acid 444 close to the C-terminus. This suggests that sequences in the PPARalpha DNA binding domain influence other receptor functions besides DNA binding.

Peroxisome proliferator-activated receptors: structures and function.

We have been attempting to elucidate the molecular mechanisms through which peroxisome proliferators exert their pleiotropic effects, with particular emphasis on understanding why humans appear unresponsive to these compounds. There is a wealth of data to implicate the peroxisome proliferator-activated receptor alpha (PPAR alpha) in mediating these effects in rodent species; PPAR alpha is expressed in tissues that show physiological changes in response to PPs, is transcriptionally activated in vitro by a variety of PPs, and it has been recently demonstrated that mice lacking this receptor are refractory to the effects of clofibrate and Wy-14,643, at least in the short term. It is conceivable that differences in PPAR alpha between responsive rodent and unresponsive human subjects may provide the key to understanding the basis of this species variation in response, and with this in mind we have been studying the biology of PPAR alpha in humans and looking at interindividual variation. There is already published evidence, albeit on only two sequences, for structural and functional polymorphism in human PPAR alphas. We have extended these findings, and shown that: There is considerable variation in hPPAR alpha cDNAs obtained from different individuals, both at the gross structural level (lack of a coding exon) and of a more subtle nature (single base changes leading to amino acid substitutions). One such cDNA, the sequence of which differs at only three amino acids from that published, encodes a receptor that is incapable of transcriptional activation by potent PPs. The degree to which hPPAR alpha transcripts are expressed in human livers can vary by up to an order of magnitude between individuals. The tissue-specific expression profile of PPAR alpha in humans is very different from that in rat and mouse. In particular, the human liver contains generally low levels of PPAR alpha in contrast to the responsive rodents, in which potent PPs cause liver tumors. Taken together, these data suggest first that human and rodent PPAR alphas differ according to a number of molecular and biochemical criteria, and secondly that there is a degree of interindividual variation in PPAR alpha structure and function. Studies are ongoing to clarify this further, but human polymorphism may go some way towards explaining the apparent paradox that active PPAR alpha receptors can be isolated from an "unresponsive" species.

Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription.

We have identified a peroxisome proliferator response element (PPRE) approx. 4300 nucleotide upstream of the rat cytochrome P-450 CYP4A1 gene. Two members of the steroid-hormone-receptor superfamily, the peroxisome proliferator-activated receptor-alpha (PPAR alpha) and the retinoid X receptor-alpha (RXR alpha), bind specifically to this element as a heterodimer, and this element confers responsiveness to the peroxisome proliferator Wyeth-14,643 when tested in co-transfection assays. A second element, located 35 nucleotides further upstream, fails to bind PPAR alpha/RXR alpha heterodimers and is unresponsive to Wy-14,643 in co-transfection assays. Both elements are, however, responsive to 9-cis-retinoic acid in the presence of RXR alpha, when tested in the co-transfection assay. As RXR alpha fails to bind to either element as a homodimer, we suggest that RXR alpha interacts with PPAR alpha to regulate transcription via the proximal element, and interacts with some other cellular factor to regulate transcription via the more distal element. This is consistent with previous reports that a number of peroxisome proliferator-regulated genes contain PPRE-like elements as part of their regulatory sequences, which may be recognized by several receptor combinations. This provides further evidence that PPARs and their co-factors are important in mediating the pleiotropic action of peroxisome proliferators.

PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene.

A DNA sequence that confers a response to a class of rodent hepatocarcinogens termed peroxisome proliferators has been identified 2947bp upstream of the rat peroxisomal bifunctional enzyme gene. Two members of the steroid hormone receptor family, termed the peroxisome proliferator activated receptor (PPAR alpha) and the retinoid X receptor (RXR alpha), co-operate to bind specifically to this sequence. Importantly, this response element (PPRE) is similar to that identified upstream of other peroxisome proliferator responsive genes such as those encoding acyl CoA oxidase and cytochrome P450 IVA6. These data therefore provide further evidence that PPAR alpha plays an important role in mediating the action of peroxisome proliferators.

Individual Xenopus histone genes are replication-independent in oocytes and replication-dependent in Xenopus or mouse somatic cells.

We have assessed the response of many histone H3 mRNAs and an H1C mRNA in Xenopus tissue culture cells after treatment with the DNA synthesis inhibitor hydroxyurea. The amount of the histone mRNAs falls rapidly in response to the inhibitor. This response is prevented by cycloheximide. Cloned Xenopus histone genes were transfected into mouse cells and a cell line was obtained in which the Xenopus genes were actively expressed giving rise to mRNA with correct 5'-termini. The Xenopus genes were correctly regulated at the level of mRNA amounts in the mouse cell line. Nuclear microinjection experiments with Xenopus oocytes and S1 nuclease analysis of normal ovary RNA showed that the H1C gene, and probably also two H3 genes, which are replication-dependent in somatic cells are expressed in oocytes and are therefore replication-independent in this cell type. The same promoters are used in both replication-dependent and independent expression.

Nucleotide sequences of H1 histone genes from Xenopus laevis. A recently diverged pair of H1 genes and an unusual H1 pseudogene.

Four clones containing H1 histone gene sequences were previously isolated from a Xenopus laevis genomic library (1) and we now present the complete nucleotide sequences of these H1 genes and their flanking regions. Two of these genes code for minor H1 proteins, probably H1C, when expressed in the oocyte transcription/translation system and are present on clones with almost identical overall organization. However, at the nucleotide level these genes differ in showing base insertions and deletions, as well as substitutions. A third gene sequence which is more related to the major X. laevis H1A, corresponds to the 3' two thirds of an H1 gene. This gene has in place of a 5' coding region at least 1800 bp of apparently noncoding sequence, some of which is A-T rich. The junction does not correspond to the consensus sequence of an intron/exon boundary and therefore this H1 sequence is more likely to represent a pseudogene. Comparisons of the coding and flanking regions of these X. laevis H1 genes indicate the kind of differences which can occur among H1 subtypes within a species. A region of homology noted in the 3' noncoding portion of vertebrate histone genes is discussed in relation to the mechanism of termination of transcription.

Organization and expression of cloned histone gene clusters from Xenopus laevis and X. borealis.

We have isolated several clones containing Xenopus histone genes from genomic libraries of X. laevis and X. borealis DNA. Each genomic clone has been mapped and the positions of 26 histone genes in seven laevis clones and 5 histone genes in one borealis clone have been determined. In laevis, the histone gene clusters show considerable variation in gene order within a single individual. When the cloned DNAs were microinjected into the nucleus of Xenopus oocytes, expression of cloned genes at the transcriptional and translational level was readily detectable. Previously unknown histone variants were revealed by the microinjection experiments.