Salmonid Pituitary Cells as a Test System for Identifying Endocrine-Disrupting Compounds.

The pituitary gland is a central regulator of reproduction, producing two gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which regulate gonadal development, sex steroid synthesis, and gamete maturation. The present study sought to optimize an in vitro test system using pituitary cells isolated from previtellogenic female coho salmon and rainbow trout, focusing on fshb and lhb subunit gene expression. Initially, we optimized culture conditions for duration and benefits of culturing with and without addition of endogenous sex steroids (17ß-estradiol [E2] or 11-ketotestosterone) or gonadotropin-releasing hormone (GnRH). The results suggest that culturing with and without E2 was valuable because it could mimic the (+) feedback effects on Lh that are observed from in vivo studies. After optimizing assay conditions, a suite of 12 contaminants and other hormones was evaluated for their effects on fshb and lhb gene expression. Each chemical was tested at four to five different concentrations up to solubility limitations in cell culture media. The results indicate that more chemicals alter lhb synthesis than fshb. The more potent chemicals were estrogens (E2 and 17α-ethynylestradiol) and the aromatizable androgen testosterone, which induced lhb. The estrogen antagonists 4-OH-tamoxifen and prochloraz decreased the E2-stimulated expression of lhb. Among several selective serotonin reuptake inhibitors tested, the sertraline metabolite norsertraline was notable for both increasing fshb synthesis and decreasing the E2 stimulation of lhb. These results indicate that diverse types of chemicals can alter gonadotropin production in fish. Furthermore, we have shown that pituitary cell culture is useful for screening chemicals with potential endocrine-disrupting activity and can support the development of quantitative adverse outcome pathways in fish. Environ Toxicol Chem 2023;42:1730-1742. © 2023 SETAC.

In vitro exposure of vitellogenic rainbow trout ovarian follicles to endocrine disrupting chemicals can alter basal estradiol-17ß production and responsiveness to a gonadotropin challenge.

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradiol-17ß (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17α-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function.

Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty.

Although estrogens have been generally considered to play a critical role in ovarian differentiation in non-mammalian vertebrates, the specific functions of estrogens during ovarian differentiation remain unclear. We isolated two mutants with premature stops in the ovarian aromatase (cyp19a1) gene from an N-ethyl-N-nitrosourea-based gene-driven mutagenesis library of the medaka, Oryzias latipes. In XX mutants, gonads first differentiated into normal ovaries containing many ovarian follicles that failed to accumulate yolk. Subsequently, ovarian tissues underwent extensive degeneration, followed by the appearance of testicular tissues on the dorsal side of ovaries. In the newly formed testicular tissue, strong expression of gsdf was detected in sox9a2-positive somatic cells surrounding germline stem cells suggesting that gsdf plays an important role in testicular differentiation during estrogen-depleted female-to-male sex reversal. We conclude that endogenous estrogens synthesized after fertilization are not essential for early ovarian differentiation but are critical for the maintenance of adult ovaries.

Regulation of sex steroid production and mRNAs encoding gonadotropin receptors and steroidogenic proteins by gonadotropins, cyclic AMP and insulin-like growth factor-I in ovarian follicles of rainbow trout (Oncorhynchus mykiss) at two stages of vitellogenesis.

At the completion of vitellogenesis, the steroid biosynthetic pathway in teleost ovarian follicles switches from estradiol-17ß (E2) to maturational progestin production, associated with decreased follicle stimulating hormone (Fsh) and increased luteinizing hormone (Lh) signaling. This study compared effects of gonadotropins, human insulin-like growth factor-I (IGF1), and cAMP/protein kinase A signaling (forskolin) on E2 production and levels of mRNAs encoding steroidogenic proteins and gonadotropin receptors using midvitellogenic (MV) and late/postvitellogenic (L/PV) ovarian follicles of rainbow trout. Fsh, Lh and forskolin, but not IGF1, increased testosterone and E2 production in MV and L/PV follicles. Fsh increased steroidogenic acute regulatory protein (star; MV), 3ß-hydroxysteroid dehydrogenase/Δ(5-4) isomerase (hsd3b; MV) and P450 aromatase (cyp19a1a; MV) transcript levels. Lh increased star mRNA levels (MV, L/PV) but reduced cyp19a1a transcripts in L/PV follicles. At both follicle stages, IGF1 reduced levels of hsd3b transcripts. In MV follicles, IGF1 decreased P450 side-chain cleavage enzyme (cyp11a1) transcripts but increased cyp19a1a transcripts. In MV follicles only, forskolin increased star and hsd3b transcripts. Forskolin reduced MV follicle cyp11a1 transcripts and reduced cyp19a1a transcripts in follicles at both stages. Fsh and Lh reduced fshr transcripts in L/PV follicles. Lh also reduced lhcgr transcripts (L/PV). IGF1 had no effect on gonadotropin receptor transcripts. Forskolin reduced MV follicle fshr transcript levels and reduced lhcgr transcripts in L/PV follicles. These results reveal hormone- and stage-specific transcriptional regulation of steroidogenic protein and gonadotropin receptor genes and suggest that the steroidogenic shift at the completion of vitellogenesis involves loss of stimulatory effects of Fsh and Igfs on cyp19a1a expression and inhibition of cyp19a1a transcription by Lh.

Automated Identification of Lesion Activity in Neovascular Age-Related Macular Degeneration.

PURPOSE: The objective of the study was to evaluate the accuracy of the Notal OCT Analyzer (NOA) versus that of a retina specialist (RS) in the automated detection of fluid on optical coherence tomography (OCT). DESIGN: A study of the performance of the NOA compared with the results from 3 RSs. PARTICIPANTS: A selection of 155 anonymized OCT scans (Zeiss Cirrus; Carl Zeiss Meditec, Dublin, CA) from an image repository at a single tertiary referral retina center (Belfast Health and Social Care Trust, Belfast, United Kingdom) after approval from the local data guardian of the clinical site. METHODS: One hundred fifty-five OCT cube scans were stripped of all clinical identifiers and exported. The NOA and 3 independent RSs analyzed all 128 B-scans of each cube scan for the presence of intraretinal fluid, subretinal fluid, and sub-retinal pigment epithelium fluid. The NOA also ranked individual B-scans of each volume scan for likelihood of CNV activity, which was subjected to a second grading session by the 3 RSs. MAIN OUTCOME MEASURES: The NOA's sensitivity and specificity versus the RS grading and the NOA's performance in ranking B-scans for activity. RESULTS: One hundred forty-two cube scans met the inclusion criteria for the primary analysis. On testing the RS grading versus the NOA, the accuracy was 91% (95% confidence interval [CI], ±7%), sensitivity was 92% (95% CI, ±6%), and specificity was 91% (95% CI, ±6%), meeting the primary outcome. The graders' accuracy when compared with the majority of the other graders (including a fourth grader) was 93%. On average, the 3 graders could identify fluid in 95% of scans by just reviewing a single cross-section with the highest NOA score and 99.5% of scans with fluid by viewing the top 3 cross-sections. CONCLUSIONS: Concordance between the NOA and the RS determination of lesion activity was extremely high. The level of discrepancy between the RS and the NOA results was similar to the NOA's mismatches. Our results show that automated delineation of the retinal contours combined with interpretation of disease activity is feasible and has the potential to become a powerful tool in terms of its clinical applications.

Tips and tricks of ureteroscopy: consensus statement. Part II. Advanced ureteroscopy.

Our "tips and tricks" focuses on all aspects of upper tract endourology and we hope these will be of use to all trainees and consultants who perform ureteroscopy. We report an "expert consensus view" from experienced endourological surgeons, on all aspects of advanced ureteroscopic techniques, with a particular focus on avoiding and getting out of trouble while performing ureteroscopy. In this paper we provide a summary of placing ureteric access sheath, flexible ureteroscopy, intra renal stone fragmentation and retrieval, maintaining visual clarity and biopsy of ureteric and pelvicalyceal tumours.

High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch.

Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain-pituitary-gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina(®) sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone ß subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone ß subunit (-3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-ß signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks, respectively, including circadian rhythm signaling, calcium signaling, peroxisome proliferator-activated receptor (PPAR) signaling, PPARα/retinoid x receptor α activation, and netrin signaling. Network analysis identified potential interactions between genes involved in circadian rhythm and GNRH signaling, suggesting possible effects of EE2 on timing of reproductive events.

Injuries from non-retention in gillnet fisheries suppress reproductive maturation in escaped fish.

Exploitation of fisheries resources has unintended consequences, not only in the bycatch and discard of non-target organisms, but also in damage to targeted fish that are injured by gear but not landed (non-retention). Delayed mortality due to non-retention represents lost reproductive potential in exploited stocks, while not contributing to harvest. Our study examined the physiological mechanisms by which delayed mortality occurs and the extent to which injuries related to disentanglement from commercial gear compromise reproductive success in spawning stocks of Pacific salmon (Oncorhynchus spp.). We found evidence for elevated stress in fish injured via non-retention in gillnet fisheries. Plasma cortisol levels correlated with the severity of disentanglement injury and were elevated in fish that developed infections related to disentanglement injuries. We also analyzed sex steroid concentrations in females (estradiol-17ß and 17,20ß-dihydroxy-4-pregnen-3-one) to determine whether non-retention impairs reproductive potential in escaped individuals. We demonstrate evidence for delayed or inhibited maturation in fish with disentanglement injuries. These findings have important implications for effective conservation and management of exploited fish stocks and suggest means to improve spawning success in such stocks if retention in commercial fisheries is improved and incidental mortality reduced.

Stage-specific effects of androgens and estradiol-17beta on the development of late primary and early secondary ovarian follicles of coho salmon (Oncorhynchus kisutch) in vitro.

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.

Disruption of the salmon reproductive endocrine axis through prolonged nutritional stress: changes in circulating hormone levels and transcripts for ovarian genes involved in steroidogenesis and apoptosis.

Mechanisms regulating the normal progression of ovarian follicular growth versus onset of atresia in fishes are poorly understood. To gain a better understanding of these processes, we exposed immature female coho salmon (Oncorhynchus kisutch) to prolonged fasting to induce follicular atresia and monitored body growth, development of the ovarian follicles, changes in reproductive hormones, and transcripts for ovarian genes. Prolonged fasting reduced body and ovary weight and increased the appearance of atretic follicles relative to normally fed controls. Endocrine analyses showed that fasting reduced plasma insulin-like growth factor 1 (IGF1), estradiol-17ß (E2), and pituitary, but not plasma, levels of follicle-stimulating hormone (FSH). Transcripts for ovarian fsh receptor (fshr) and steroidogenesis-related genes, such as steroidogenic acute regulatory protein (star), 3ß-hydroxysteroid dehydrogenase (hsd3b), and P450 aromatase (cyp19a1a) were significantly lower in fasted fish. Ovarian expression of apoptosis-related genes, such as Fas-associated death domain (fadd), caspase 8 (casp8), caspase 3 (casp3), and caspase 9 (casp9) were significantly elevated in fasted fish compared to fed fish, indicating that apoptosis is involved in the process of atresia in this species. Interestingly, some genes such as fadd, casp8, casp3, and hsd3b, were differentially expressed prior to increases in the number of atretic follicles and reductions in hormone levels induced by fasting, and may therefore have potential as early indicators of atresia. Together these results suggest that prolonged nutritional stress may disrupt the reproductive system and induce follicular atresia in part via reductions in ovarian IGF and FSH signaling, and downstream effects on steroidogenesis-related genes and E2 production.

Predictive response-relevant clustering of expression data provides insights into disease processes.

This article describes and illustrates a novel method of microarray data analysis that couples model-based clustering and binary classification to form clusters of `response-relevant' genes; that is, genes that are informative when discriminating between the different values of the response. Predictions are subsequently made using an appropriate statistical summary of each gene cluster, which we call the `meta-covariate' representation of the cluster, in a probit regression model. We first illustrate this method by analysing a leukaemia expression dataset, before focusing closely on the meta-covariate analysis of a renal gene expression dataset in a rat model of salt-sensitive hypertension. We explore the biological insights provided by our analysis of these data. In particular, we identify a highly influential cluster of 13 genes--including three transcription factors (Arntl, Bhlhe41 and Npas2)-that is implicated as being protective against hypertension in response to increased dietary sodium. Functional and canonical pathway analysis of this cluster using Ingenuity Pathway Analysis implicated transcriptional activation and circadian rhythm signalling, respectively. Although we illustrate our method using only expression data, the method is applicable to any high-dimensional datasets. Expression data are available at ArrayExpress (accession number E-MEXP-2514) and code is available at http://www.dcs.gla.ac.uk/inference/metacovariateanalysis/.

Targeting chronic myeloid leukemia stem cells.

Chronic myeloid leukemia (CML) arises as a consequence of a chromosomal translocation giving rise to the Philadelphia chromosome and Bcr-Abl oncogene. CML is a clonal disease of stem cell origin and an excellent example of a malignancy in which tumor-initiating cells may hold the key to disease eradication. The known molecular basis of CML has enabled the development of Abl-specific tyrosine kinase inhibitors, such as imatinib mesylate. However, the success of tyrosine kinase inhibitors, as rationally designed first-line therapies, has been tempered by problems of disease persistence and resistance. Residual disease has been shown to be enriched within the stem cell compartment and to persist at stable levels for up to 5 years of complete cytogenetic response. This finding has led to further searches for novel strategies aimed at eliminating these cells; such strategies may be essential in achieving cure. The most significant recent findings are discussed in this review.

Differential suppressive effects of low physiological doses of estradiol-17beta in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in previtellogenic ovarian follicles of rainbow trout.

Numerous recent reports have demonstrated effects of estrogenic chemicals on reproductive physiology of fish. However, there is little information available on the regulation of ovarian steroidogenesis by physiological levels of endogenous steroids in teleosts. Therefore, we analyzed the levels of mRNAs encoding steroidogenic proteins in ovaries of E2-treated rainbow trout Oncorhynchus mykiss). Previtellogenic (perinucleolar oocyte stage) trout received either blank or E2 implants (0.1 microg, 1 microg or 10 microg/g BW) for 7 days in order to achieve low, medium and high physiological levels of E2 in plasma. Plasma E2 levels were measured using radioimmunoassay. Levels of mRNAs encoding steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450 aromatase A (P450aromA) in the ovary were analyzed by real-time quantitative PCR. E2 levels in control animals were approximately 0.5 ng/ml. Levels in treated fish were approximately 1 ng/ml (0.1 microg implant), 2.6 ng/ml (1 microg implant) and 90 ng/ml (10 microg implant), within or just above the physiological range of immature and maturing female rainbow trout. StAR mRNA levels were significantly reduced by all E2 treatments. P450scc mRNA levels were not affected, but 3beta-HSD and P450arom mRNA levels were significantly decreased by the 1 and 10 microg E2/BW implants. These results indicate that E2, either directly or indirectly, downregulates expression of StAR and major steroidogenic enzyme genes in rainbow trout ovary. Furthermore, expression of the trout StAR gene seems particularly sensitive to E2.

Unilateral ovariectomy increases egg size and reduces follicular atresia in the semelparous coho salmon, Oncorhynchus kisutch.

Unilateral ovariectomy (ULO, removal of one ovary) is a powerful technique for studying aspects of reproductive physiology, including follicular recruitment and growth. To examine effects of ULO for the first time in a semelparous species, coho salmon (Oncorhynchus kisutch) were unilaterally ovariectomized during mid-vitellogenesis approximately 3 months before spawning. At termination of the study (79 days post-surgery), single ovaries of ULO fish were gravimetrically equivalent to paired ovaries of sham surgery, control fish. There was no evidence of recruitment of new vitellogenic follicles. Instead, the dramatic increase in ovary mass was attributable to hypertrophy of existing vitellogenic follicles (33% increase in volume) and increased fecundity achieved through a greater than two-fold reduction in follicular atresia. The composition of whole ovaries on a dry weight basis from ULO fish was greater in protein, but lower in lipid than that of control fish. Expressing the data on a per follicle basis, however, showed that follicles of ULO fish contained more protein, ash, water, and lipid. The results indicate that ULO of coho salmon induces compensatory hypertrophy of existing vitellogenic follicles, while maximizing fecundity through reduction of atresia. Thus, 3 months before spawning, coho salmon exhibit the ability to adjust final egg size and number when faced with significant depletion of ovarian follicles. This in vivo system provides a platform for further study of physiological mechanisms regulating follicular growth and atresia, and the trade-off between egg size and egg number.

11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro.

Previtellogenic ovarian fragments from eel, Anguilla australis, were cultured in vitro in a chemically defined medium containing steroids and/or peptide hormones for 18 days in order to investigate their involvement in control of early oocyte growth. 11-Ketotestosterone (11-KT), but not estradiol-17beta, induced a significant 10-20% increase in diameters of previtellogenic oocytes and oocyte nuclei in a dose-dependent manner. Effects were greatest for 100 nM 11-KT, a dose that is within the physiological range seen in very early vitellogenic eels in the wild. The effect was not accompanied by obvious ultrastructural changes in the oocytes other than an apparent increase in nuclear size. Similarly, treatment with recombinant human IGF-I resulted in increased oocyte diameters, whereas no such effect was seen after treatment with heterologous insulin, GH, leptin, or human chorionic gonadotropin. Interestingly, lipid supplementation also resulted in an increase in oocyte diameter, and greater radioactivity in ovarian explants following incubation with (14)C-triglycerides and 11-KT, but not FSH, suggesting that the androgen may play a role in lipid accumulation into the oocyte. Our results implicate hormones from both the reproductive and the metabolic axes in control of previtellogenic oocyte growth in a teleost fish.

Effects of artificial social stimuli on the reproductive schedule and hormone levels of yellow-eyed penguins (Megadyptes antipodes).

The effects of social stimuli on avian reproductive behaviors such as breeding schedules and courtship behaviors are well known due to numerous field studies. However, studies that have simultaneously examined the effects of social stimuli on reproductive behavior and the mediating endocrine mechanisms have been largely restricted to captive populations, which may not be representative of free-living populations. This study, conducted over two breeding seasons, aimed to simultaneously measure the effects of experimentally increasing auditory stimuli on the breeding schedule and endocrinology (levels of total androgen, estradiol, progesterone and prolactin) on free-living yellow-eyed penguins (Megadyptes antipodes). The yellow-eyed penguin is the least colonial of all penguins, nesting far apart from each other under dense vegetation, and, therefore, is presumed to experience much lower levels of social stimuli than other penguins. Egg laying was significantly more synchronous and tended to be earlier when birds were exposed to playbacks of the calls of conspecifics in 1 year of the study. We also found that levels of total androgen and estradiol of males in 1 year, and prolactin in another year, were proportionally higher among treated birds compared control birds that received no artificial auditory stimuli. These results show that even among supposedly solitary nesters, social stimuli could still play a role in influencing reproductive behavior and physiology. For the first time in free-living seabirds, we have demonstrated that behavioral responses to increased social stimuli are associated with hormonal changes.

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell-capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93.9% (495/527 patients) for peripheral blood and 97.6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 x 10(9) cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.

Transformation and progression of Waldenström's macroglobulinemia following cladribine therapy in two cases: natural evolution or iatrogenic causation?

We report two cases of Waldenström's macroglobulinemia with an unusual aggressive transformation following treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA), a purine analogue. The first patient developed transformation to a diffuse large-cell non-Hodgkin lymphoma, while the second developed extensive extramedullary involvement. Both patients displayed rapid progression following transformation and were refractory to chemotherapy. Both patients were pretreated with multiple courses of prednisone and chlorambucil, and transformation occurred shortly after therapy with cladribine. We propose that immune suppression from alkylating agents and purine analogues may have contributed to the unusual progression, resulting in a dismal outcome.

Effects of ACTH and cAMP on steroidogenic acute regulatory protein and P450 11beta-hydroxylase messenger RNAs in rainbow trout interrenal cells: relationship with in vitro cortisol production.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane, thereby making the molecule available for cholesterol side-chain cleavage enzyme, which carries out the first conversion in the steroidogenic pathway. In mammals, StAR controls this rate limiting step in steroidogenesis, and both StAR protein and StAR mRNA levels become rapidly elevated in response to tropic hormone stimulation. The relationship between StAR gene expression and steroid production in fish has not yet been well explored. We investigated the relationship between adrenocorticotropic hormone (ACTH)- and cAMP-stimulated cortisol production in vitro and levels of StAR transcripts in interrenal cells of rainbow trout. To assess the effect of ACTH on mRNA levels of a downstream steroidogenic enzyme, we also investigated the effects of ACTH on transcripts encoding 11beta hydroxylase (P450 11beta). In a series of experiments, juvenile rainbow trout head kidney tissue containing interrenal cells was incubated with either ACTH or dibutyryl cyclic AMP (dbcAMP). Cortisol in incubation media were measured by radioimmunoassay and total RNA was isolated from the tissue for Northern analysis or for quantitative real-time PCR. Incubation of tissue with 150 ng/mL ACTH for 1-18 h induced a progressive increase in cortisol accumulation in media, but StAR mRNA levels increased modestly and mostly insignificantly over 18 h, irrespective of treatment. Exposure of tissue for 18 h to 5, 150, 500 or 1,500 ng ACTH/mL resulted in a strong increase in cortisol production, with a peak response (15-fold increase over controls) achieved with 150 ng/mL ACTH. Although there was a trend towards a dose-response effect, mean StAR mRNA levels were only significantly affected by the highest concentration of ACTH used (1,500 ng/mL), which induced a less than 2-fold increase in StAR transcripts. However, there was a significant linear relationship between StAR mRNA levels and ACTH-induced cortisol accumulation in media (p<0.001, r(2)=0.55). Incubation of tissue with 5mM dbcAMP for 6 or 18 h induced large increases in cortisol accumulation in media over controls, but had no significant effect on StAR mRNA levels. By contrast, ACTH induced a clear dose-dependent increase in P450 11beta transcripts, with 150 ng/mL ACTH inducing an 8-fold increase in levels compared to control; nonetheless, only a weak correlation existed between transcript levels and ACTH-induced cortisol secretion (p<0.003, r(2)=0.26). Thus, despite the relatively high degree of conservation of StAR proteins in vertebrates, we have been unable to demonstrate that a rapid, acute increase in transcription of the StAR gene is the dominant mechanism supporting flow of cholesterol to the mitochondria during acute increases in cortisol production in rainbow trout. The strong stimulation of P450 11beta gene transcription by ACTH probably enhances biosynthetic capacity during longer term chronic ACTH stimulation.