Andrew S. Friedmann

Low levels of dietary methylmercury inhibit growth and gonadal development in juvenile walleye (Stizostedion vitreum)

Mercury levels in the aquatic environment of North America have been increasing, raising the possibility that this highly toxic heavy metal might alter fish populations. Previous investigations have demonstrated toxic effects of mercury on teleost reproduction, but these findings were observed following unrealistically high exposures. In this study, we used concentrations frequently observed in North American lakes to investigate the effects of dietary methylmercury on growth, gonadal development, and plasma cortisol levels in juvenile walleye (Stizostedion vitreum). For a period of 6 months, two groups of walleye were reared on untainted catfish fillets, while two test groups were fed fillets injected with methylmercury, one group receiving 0.1 kg Hg g-’ food (low-mercury diet) and the other receiving 1 .O 1.18 Hg g 1 food (high-mercury diet). After the exposure period, fish fed the low- and high-mercury diets had mean body burdens of 0.254 f. 0.015 pg Hg g-l and 2.37 f 0.09 Fg Hg g-‘, respectively. Dietary mercury significantly impaired both growth and gonadal development in males, which was apparent as reduced fish length, weight, and gonadosomatic index. Testicular atrophy was observed in fish fed the mercury-tainted fillets, but was nonexistent in control animals. Mercury also suppressed plasma cortisol in juveniles (sexes combined). The findings of this study suggest that dietary methylmercury, at levels currently found in the aquatic environment, might reduce juvenile survival by impairing growth and immune function. Furthermore, these results suggest that methylmercury might also affect reproductive potential of teleosts by impairing testicular development in young.

Vasopressin gene related products are markers of human breast cancer

SummaryImmunohistochemical analysis for products of vasopressin and oxytocin gene expression was performed on acetone-fixed tissues from 19 breast cancers representing a variety of tumor sub-types. Studies employed the avidin-biotin complex (ABC) immunohistochemical procedure and utilized rabbit polyclonal antibodies to arginine vasopressin (VP), provasopressin (ProVP), vasopressin-associated human glycopeptide (VAG), oxytocin (OT), oxytocin-associated human neurophysin (OT-HNP), and a mouse monoclonal antibody to vasopressin-associated human neurophysin (VP-HNP). Western Blot analysis was performed on protein extracts of fresh-frozen tissues from 12 additional breast tumors. While VP gene related proteins were not detected in normal breast tissue, immunohistochemistry revealed the presence of VP, Pro VP, and VAG in all neoplastic cells for all of the tumor tissues examined. Vasopressin-associated human neurophysin was evident in only one of 19 acetone-fixed tumor preparations. However, Western blot analysis for all 12 fresh-frozen tumor samples showed the presence of two proteins, 42,000 and 20,000 daltons, that were immunoreactive with antibodies to VP, VP-HNP, and VAG. Oxytocin and OT-HNP, by immunohistochemistry, were found to be common to cells of normal breast tissues. For tumors, positive staining for OT was observed in 8 of 18 tumors, while OT-HNP was not detected in any of the tumors examined. These findings indicate that VP gene expression is a selective feature of all breast cancers, and that products of this expression might therefore be useful as markers for early detection of this disease and as possible targets for immunotherapy.

Products of vasopressin gene expression in small-cell carcinoma of the lung.

Small-cell neuroendocrine carcinoma of the lung is known to express products related to the vasopressin gene, although these products have been reported to sometimes differ from those generated by neurones of the hypothalamo-neurohypophyseal system. To further investigate vasopressin gene expression in neuroendocrine carcinomas, we performed immunohistochemistry on 24 histologically classified small-cell carcinomas using antibodies directed against different regions of the vasopressin precursor. All of the tumours examined contained at least two parts of the vasopressin precursor, suggesting that vasopressin might have a biological role in these tumours and indicating a role for these products in tumour diagnosis and treatment. Sixty-seven per cent of the tumours contained immunoreactivity for all major regions of the precursor: vasopressin, vasopressin-associated human neurophysin, the bridging region between the hormone and the neurophysin, and vasopressin-associated human glycopeptide. However, 33% of the tumours examined appeared to express only part of the vasopressin precursor, as evidenced by the absence of immunoreactivity for the neurophysin and/or the glycopeptide. These results support the proposition that both normal and abnormal vasopressin gene expression occurs in small-cell carcinoma of the lung.

Vasopressin and oxytocin production by non-neuroendocrine lung carcinomas: an apparent low incidence of gene expression

In previous studies we have demonstrated the high incidence of vasopressin gene expression as a characteristic feature of small-cell carcinoma of the lung. In the present study we examined expression of this gene in non-neuroendocrine tumors to determine if vasopressin production is a common feature of all lung tumors. We carried out the immunohistochemical evaluation of 22 non-neuroendocrine tumors (12 adenocarcinomas and 10 squamous-cell carcinomas) with antibodies to vasopressin, to oxytocin, and to their related neurophysins. The antibody preparations directed against vasopressin, oxytocin, or oxytocin-associated human neurophysin did not react with any of the tumors examined. Of two monoclonal antibodies to vasopressin-associated human neurophysin used, one did not react with any of the tumors, while the other stained neoplastic cells in only one adenocarcinoma and one squamous-cell carcinoma. These findings, taken with previous reports, indicate that among lung carcinomas, a high incidence of vasopressin/oxytocin gene expression is confined to neuroendocrine tumors.

Factors regulating the production of vasopressin-associated human neurophysin by small-cell carcinoma of the lung: Evaluation by computer-enhanced quantitative immunocytochemistry

Expression of the vasopressin gene appears to be a property common to all small-cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin-associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br-cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production.

Vasopressin and vasopressin-receptor immunoreactivity in small-cell lung carcinoma (SCCL) cell lines: disruption in the activation cascade of V1a-receptors in variant SCCL

Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V1a-receptor immunoreactivity. One of these classical cell lines, NCI-H345, and one variant cell line, NCI-H82, were further investigated for binding of V1 and V2 vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCI-H345 demonstrated similar binding patterns with the V1 and V2 vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 microM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCI-H82). However, vasopressin (0.1 or 1 microM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V1a and V2 receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V1a receptors as indicated by the lack of vasopressin-induced calcium mobilization.

Biosynthesis of vasopressin by gastrointestinal cells of Brattleboro and Long-Evans rats

The distribution of vasopressin, provasopressin, vasopressin-associated neurophysin, and vasopressin-associated glycopeptide was determined immunohistochemically in the gastrointestinal system of Brattleboro and Long-Evans rats. Cells containing immunoreactivity for vasopressin, provasopressin, neurophysin, and glycopeptide were detected in the same cell types of the stomach and duodenum, while selected cells in the duodenum contained only immunoreactive glycopeptide. Unlike that in the hypothalamus, staining for neurophysin in the gastrointestinal tract was sensitive to fixation. These findings indicate that vasopressin is produced by cells in the rat gastrointestinal system and suggest the existence of synthetic pathways different from those found in hypothalamic neurons.

Evidence for vasopressin production in the human gastrointestinal system

In the present study we performed immunohistochemical examination of segments from the human gastrointestinal system for the presence of cells containing vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP). VP immunoreactivity was found in crypt cells of the stomach and small intestine, and in mononuclear cells within the lamina propria and submucosa. VP-HNP was demonstrated in the crypt and lamina propria regions of the small intestine, and was colocalized with vasopressin in crypt cells. This colocalization indicates local vasopressin synthesis by these cells and raises the possibility that they may perform an endocrine or exocrine function in the human gastrointestinal system.