Molecular and cellular effects of chemicals disrupting steroidogenesis during early ovarian development of brown trout (Salmo trutta fario).

A range of chemicals found in the aquatic environment have the potential to influence endocrine function and affect sexual development by mimicking or antagonizing the effects of hormones, or by altering the synthesis and metabolism of hormones. The aim of this study was to evaluate whether the effects of chemicals interfering with sex hormone synthesis may affect the regulation of early ovarian development via the modulation of sex steroid and insulin-like growth factor (IGF) systems. To this end, ex vivo ovary cultures of juvenile brown trout (Salmo trutta fario) were exposed for 2 days to either 1,4,6-androstatriene-3,17-dione (ATD, a specific aromatase inhibitor), prochloraz (an imidazole fungicide), or tributyltin (TBT, a persistent organic pollutant). Further, juvenile female brown trout were exposed in vivo for 2 days to prochloraz or TBT. The ex vivo and in vivo ovarian gene expression of the aromatase (CYP19), responsible for estrogen production, and of IGF1 and 2 were compared. Moreover, 17ß-estradiol (E2) and testosterone (T) production from ex vivo ovary cultures was assessed. Ex vivo exposure to ATD inhibited ovarian E2 synthesis, while T levels accumulated. However, ATD did not affect ex vivo expression of cyp19, igf1, or igf2. Ex vivo exposure to prochloraz inhibited ovarian E2 production, but did not affect T levels. Further prochloraz up-regulated igf1 expression in both ex vivo and in vivo exposures. TBT exposure did not modify ex vivo synthesis of either E2 or T. However, in vivo exposure to TBT down-regulated igf2 expression. The results indicate that ovarian inhibition of E2 production in juvenile brown trout might not directly affect cyp19 and igf gene expression. Thus, we suggest that the test chemicals may interfere with both sex steroid and IGF systems in an independent manner, and based on published literature, potentially lead to endocrine dysfunction and altered sexual development.

Development of an ex vivo brown trout (Salmo trutta fario) gonad culture for assessing chemical effects on steroidogenesis.

A variety of natural and synthetic environmental substances have been shown to disrupt vertebrate reproduction through mimicking or modifying the regulation of the endocrine system. Tests to screen for any such chemicals that directly interact with the steroid hormone receptors are widely available; however, few tests have been developed to identify chemicals that affect endocrine function through non-receptor mediated mechanisms. The aim of this study was, therefore, to develop an assay for the identification of substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis cascade, in particular the aromatase enzyme, CYP19, that catalyses the final conversion of androgens to estrogens. A gonad ex vivo assay was developed using gonad explants harvested from juvenile brown trout and cultured in a modified Leibovitz medium. Effects on sex steroid biosynthesis were quantified through measurement of 17ß-estradiol (E2) and testosterone (T) concentrations in the medium after 2 days incubation. Exposure of ovary explants to 100 ng/mL 1,4,6-androstatriene-3,17-dione (ATD), a potent pharmaceutical aromatase inhibitor, reduced E2 concentrations and elevated T concentrations confirming that CYP19 activity could be inhibited in the assay. Exposure of ovary explants to 250 ng/mL prochloraz, an imidazole fungicide, also reduced E2 concentrations but did not affect T levels, consistent with reports that in addition to inhibiting CYP19 activity, prochloraz also inhibits enzymes in the steroidogenic pathway upstream of the CYP19 enzyme. Exposure to a third chemical, tributyltin (TBT), did not affect T or E2 concentrations, further supporting previous evidence that the CYP19 modulating effects of this chemical are not mediated through direct inhibition of CYP19 activity. These results demonstrate that the gonad ex vivo assay developed here can be successfully used to identify substances that disrupt sex steroid biosynthesis and further that it has the potential to inform on their specific mode of action.

Assessing the biological potency of binary mixtures of environmental estrogens using vitellogenin induction in juvenile rainbow trout (Oncorhynchus mykiss).

Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as the endpoint. The estrogenic potencies of estradiol-17beta (E2), 4-tertnonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L(-1) for E2, 6.1 and 6.4 microg L(-1) for NP, and 4.4 and 6.5 microg L(-1) for MXC. Concentration-response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor alpha agonist and an estrogen receptor beta antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range forwhich the expected response curve forthe binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.

Detection of polymorphism in the RING3 gene by high-throughput fluorescent SSCP analysis.

We describe the use of a high-throughput, fluorescent, polymorphism-detection system, based on single-strand conformation polymorphism to screen for polymorphism in the RING3 gene. This is the first extensive mutation screen of this major histocompatibility complex-linked gene, and the entire coding region and intron-exon junctions were examined by multiplexing over 3000 polymerase chain reaction products. These techniques should be applicable for analysis of variation in other human genes. Investigation of DNA from acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) patients, as well as healthy individuals revealed low levels of polymorphism across the RING3 gene. Comparison of the distribution of genotypes at each polymorphic site between patients and healthy individuals revealed a single site which significantly deviates from Hardy-Weinberg proportions.

Chromosomal localization, gene structure and transcription pattern of the ORFX gene, a homologue of the MHC-linked RING3 gene.

We have mapped the human ORFX gene to chromosome 9q34 and determined its complete gene structure. Comparison with RING3, the human MHC-linked homologue on 6p21.3, shows the two gene structures to be highly conserved but with an approximate threefold expansion in the ORFX introns. RING3 and ORFX are found to be ubiquitously expressed in human adult and foetal tissues. Evidence suggests that the two genes may have arisen from an ancient duplication in a common ancestral chromosome.

The chromosome 6 sequencing project at the Sanger Centre.

Chromosome 6 is probably best known for encoding the major histocompatibility complex (MHC) which is essential to the human immune response. In addition, it has been shown to be associated with many diseases such Schizophrenia, Diabetes, Arthritis, Haemochromatosis, Narcolepsy, Epilepsy, Retinitis Pigmentosa, Deafness, Ovarian Cancer, and many more. Chromosome 6 is about 180 Mb in size and is estimated to encode around 3500 genes of which only about 10% are currently known. It is our aim to map, sequence and annotate the entire chromosome in close collaboration with the chromosome 6 community.

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).