Gene Expression Changes Related to Endocrine Function and Decline in Reproduction in Fathead Minnow (Pimephales promelas) after Dietary Methylmercury Exposure

Background Methylmercury (MeHg) is a known neurotoxic agent, but the mechanisms by which MeHg may act on reproductive pathways are relatively unknown. Several studies have indicated potential changes in hormone levels as well as declines in vertebrates with increasing dietary MeHg exposure. Objectives The purpose of this study was to identify alterations in gene expression associated with MeHg exposure, specifically those associated with previously observed changes in reproduction and reproductive biomarkers. Fathead minnows, Pimephales promelas, were fed one of three diets that were similar to documented concentrations of MeHg in the diets of wild invertivorous and piscivorous fish. We used a commercial macroarray in conjunction with quantitative polymerase chain reaction to examine gene expression in fish in relation to exposure to these environmentally relevant doses of MeHg. Results Expression of genes commonly associated with endocrine disruption was altered with Hg exposure. Specifically, we observed a marked up-regulation in vitellogenin mRNA in individual Hg-exposed males and a significant decline in vitellogenin gene expression in female fish with increasing Hg concentrations. Other genes identified by the macroarray experiment included those associated with egg fertilization and development, sugar metabolism, apoptosis, and electron transport. We also observed differences in expression patterns between male and female fish not related to genes specifically associated with reproduction, indicating a potential physiological difference in the reaction of males and females to MeHg. Conclusion Gene expression data may provide insight into the mechanisms by which MeHg affects reproduction in fish and indicate how MeHg differs in its effect from other heavy metals and endocrine-disrupting compounds.

The p53 tumour-suppressor gene encodes a 53 kDa nuclear phosphoprotein which is thought to protect cells against the accumulation of genetic alterations. Overexpression of the wild-type (wt) p53 protein and an increase in transcriptional transactivation activity, following treatment with DNAdamaging agents, lead to cell cycle arrest in the GI phase or the induction of apoptosis. (Vogelstein & Kinzler, 1992;Lane, 1993;Levine et al., 1994). Abnormalities in the p53 gene are the most common genetic alteration in human cancer (Hollstein et al., 1991;Levine et al., 1991;Caron de Fromentel & Soussi, 1992). In normal tissue wt p53 protein is difficult to detect, whereas in cells with p53 gene mutations, conformational changes and a prolonged biological half-life lead to accumulation of mutant p53 protein.
In human pancreatic cancer, genomic alterations in the p53 tumour-suppressor gene are frequently combined with mutations in the c-K-ras oncogene. Summarising the data from six groups (Barton et al., 1991;Ruggeri et al., 1992;Casey et al., 1993;Kalthoff et al., 1993;Scarpa et al., 1993; Perucho, personal communication) the mutation pattern of the p53 gene in pancreatic cancer shows a similar distribution to other gastrointestinal adenocarcinomas, with hotspots at positions 273,248,175 and additionally at positions 220 and 132.
Alterations of p53 cannot only be detected with molecular biological and immunohistochemical methods. In addition, mutant p53 proteins may serve as targets of the host immune system as tumour-specific antigens (Harris & Hollstein, 1993). Several previous studies have described the detection of antibodies against p53 protein in the sera of patients with various malignant diseases (Crawford et al., 1982(Crawford et al., , 1984Caron de Fromentel et al., 1987;Davidoff et al., 1992;Hassapoglidou & Diamindis, 1992;Schlichtholz et al., 1992;Winter et al., 1992;Volkmann et al., 1993).
The aim of this study was to investigate whether p53specific antibodies could be found in the sera of patients with malignant and benign pancreatic diseases, since in previous analyses (Kalthoff et al., 1993) we were able to show specific p53 immunoreactivity in cytospin preparations derived from patients with pancreatic cancer and, in addition, in specimens from patients with acute and chronic pancreatitis.
The analysis of 160 sera was performed using two independently developed ELISA systems. To further substantiate the results, all sera were analysed by Western blot technique using a cell lysate of PancTu-l cells (p53 mutation Correspondence: H. Kalthoff. Received 25 May 1994; and in revised form 12 July 1994. at codon 176) as the antigen source. Additionally, all positive sera were analysed by Western blot technique using recombinant p53 as the antigen source. We found p53 antibodies in the sera of 5/78 pancreatic tumour patients and in the sera 2/,82 of patients with benign pancreatic diseases. Two other specimens from this group tested positive, one with only weak reactivity and the other derived from a patient suffering from a squamous epithelial carcinoma of the tongue in addition to having chronic pancreatitis.

Materiais and methoi
Patient groups Serum samples from 78 patients with pancreatic cancer were analysed. In 60 patients a tissue diagnosis was obtained. In 57 patients with a malignant tumour the diagnosis was histologically proven adenocarcinoma; three patients had endocrine tumours. Tissue samples were not available in some patients (18) with inoperable cancer who underwent either palliative surgery or endoscopic drainage.
Serum samples of 82 patients with benign pancreatic diseases were analysed in this study. Of these 37 had chronic pancreatitis. The diagnosis of chronic pancreatitis was based on the presence of calcifications, pseudocysts, stenosis or destruction of the pancreatic duct at endoscopic retrograde cholangiopancreatography (ERCP). In 27 patients the pancreatitis was not a first-time event, but no gross structural changes of the pancreas were recorded in these cases. The diagnosis was based on typical clinical signs such as case history, symptoms, elevation of amylase and lipase. Eighteen of the patients had an acute pancreatitis and no medical history of previous pancreatic diseases.

ELISA systems
An anti-p53 autoantibody sandwich ELISA with solid-phase recombinant p53 protein was purchased from Dianova (Hamburg, Germany) and the analyses of the sera performed according to the manufacturer's recommendations. All sera were analysed in a second ELISA system kindly provided by T. Soussi, Paris (Schlichtholz et al., 1994).

Solubilisation
Cells were washed once with ice-cold phosphate-buffered saline (PBS). Subsequently, cells were washed twice with cell wash buffer [ice-cold PBS, containing 1.5 mM EDTA, 100 liM phenyl methyl sulphonyl fluoride (PMSF), 1 gLg ml-' aprotinin (Trasylol), adjusted to pH 7.4] and then resuspended in cell wash buffer. The cell suspension was centrifuged for 4 min at 1,400 r.p.m. and the cell pellet resuspended in lysis buffer [10 mM TRIS, containing 1.5 mM EDTA, 100 im PMSF, 1 igml1l aprotinin (Trasylol) adjusted to pH 7.4]. The cell suspension was incubated for 5 min at 4°C with occasional gentle mixing, followed by the addition of 20 gil of AEA (Antigen Extraction Agent, Oncogene Science, Dianova) for every 100 il of cell suspension, with further incubation for 5 min at 4'C and occasional vortexing. The extracts were transferred to microcentrifuge tubes and centrifuged at 4°C for 15 min at 14,000 r.p.m. The supernatant was collected and retained for protein determination and Western blotting.
The protein content of the cellular extracts was measured by the BCA Protein Assay (Pierce, Rockford, IL, USA).
Western blot PancTu-l cell lysate and recombinant wt p53 protein, kindly provided by W. Deppert (HPI, Hamburg, Germany), were electrophoretically separated by SDS-PAGE in a 10% gel and subsequently transferred to nitrocellulose filters.
After blotting, the nitrocellulose sheets were blocked in 5% bovine serum albumin (BSA) in PBS for 3 h at room temperature. The sheets were washed three times for 5 min with washing buffer (PBS, 0.05% Tween 20). Strips were incubated with the primary antibodies for 2 h and 30 min at room temperature on a rocking platform. As positive controls we used MAb 1801 (Oncogene Science, Dianova) and PAb HSP 53/2 (IgG fraction of a rabbit, hyperimmunised with recombinant wt p53 protein). All serum samples were diluted 1:100 in PBS containing 5% BSA. Following the first incubation, the strips were washed three times for 5 min with washing buffer (PBS, 0.05% Tween 20). As secondary antibodiescorresponding to the first antibodieswe used a peroxidase-conjugated AffiniPure F(ab')2 fragment of goat anti-human IgA + IgG + IgM (H + L), a peroxidaseconjugated AffiniPure F(ab% fragment of goat anti-mouse (H + L) and a peroxidase-conjugated AffiniPure goat antirabbit IgG F(ab% fragment (all from Jackson Immunoresearch Laboratories, USA), which were diluted 1:1,000 in PBS containing 5% BSA. Incubation was performed on a rocking platform for 1 h at room temperature. The subsequent washing steps were performed as described above. To visualise the reaction, the strips were incubated in a reagent comprising 4 ml of 4-chloro-1-naphthol [0.3% (w/v) in methanol] and 20 ml of substrate buffer (20 mM) Tris pH 7.4, 150 mM sodium chloride) and 6 gil of hydrogen peroxide (30%, v/v). The reaction was stopped with distilled water.

Results
In both the independently developed ELISA systems, 5 of 78 serum samples from patients with pancreatic cancer were positive for anti-p53 antibodies (Table Ia). Specimens which were positive by the ELISA methods were confirmed by the Western blot techniques using PancTu-I cell lysate ( Figure 1) and recombinant wt p53 protein ( Figure 2) as antigen sources. In all five patients the tumour had metastasised into the liver. In two of the patients a histological tissue diagnosis was available and showed an adenocarcinoma. Four of the patients were males. The age ranged from 45 to 72 years with a median of 59.4 years. All five patients had high CA 19/9 levels and three patients had high CEA levels. All five patients died within 7 months after the pancreatic cancer was diagnosed (the survival time ranging from 2 to 7 months).
Seventy-three serum samples from patients with pancreatic cancer were negative in both ELISA systems and confirmed by Western blot technique. In 67 of the 73 p53 antibodynegative patients with pancreatic cancer the tumours had metastasised into the liver as well. In 58 of the patients a tissue diagnosis was obtained fifty-five were classified as adenocarcinomas and three as endocrine tumours. Fifty-two patients were males. The age ranged from 40 to 77 years with a median of 60.4 years. Sixty-seven patients had either high CEA or high CA 19/9 levels or both (data not shown).
In the group of patients with benign pancreatic disease, one patient with chronic and one with actue pancreatitis were positive for p53 antibodies (Table lb). One patient (serum no. 2,360) with acute pancreatitis and no previous history of pancreatic disease tested positive in the ELISA system from Dianova, but not in the other ELISA system. Both Western blot techniques showed a clear reaction (Figures I and 2). At the time of serum sample collection this patient was suspected of having autoimmune hepatitis. One patient (serum no. 2,722) with acute pancreatitis, a prior history of pancreatitis and no signs of malignancy on ERCP or endoscopic ultrasonography, and who died from septic shock 1 month after admission to hospital, was positive in both ELISA systems (Table lb). A reaction was seen with the Western blot technique using PancTu-I cell lysate as the antigen source and was confirmed by a strong signal in the Western blot with the recombinant wt p53 (Figures 1 and 2). One patient (serum no. 1,033) with a chronic pancreatitis was positive in both ELISA systems and showed strong reactions in both Western blot systems (Table Tb, Figures 1 and 2). This patient had a squamous epithelial carcinoma of the tongue at the time of sample collection. Since the squamous epithelial carcinoma as well as the chronic pancreatitis could have been the reason for the anti-p53 positivity the patient's data are listed separately in Table lb. In addition to the three patients described above, one patient (sera nos. 1,134 and 1,154) had a chronic pancreatitis and no malignancy or any other disease at the time of serum collection. A resection of the pancreatic head (partial duodenopancreatectomie) was performed 6 months later and histology only revealed characteristics of an advanced chronic pancreatitis. No signs of malignancy were recorded. The serum samples were positive in the Dianova ELISA system, but showed no clear reaction in the other ELISA system. No reaction was seen with the Western blot technique using the PancTu-l cell lysate, but a faint reaction was seen with the recombinant p53 (Table lb, Figures 1 and   2).

Discossdo
The results of the two independently developed ELISA systems for the detection of antibodies against p53 correlated well, both with each other and with the Western blot analysis using PancTu-l cell lysate and recombinant wt p53 as the antigen source.
In an analysis of 790 serum samples from patients with various malignancies, only 16 positive samples (2%) were identified by Hassapoglidou and Diamandis (1992). The prevalence of p53 antibodies in the patients with pancreatic cancer in our study is lower than in serum samples of patients with colorectal cancer (Crawford et al., 1984), despite the fact that the rate of p53 gene mutations in colorectal and pancreatic cancer is very similar, suggesting either a particular mutation pattern in pancreatic cancer with low immunogenicity or a generally suppressed immune system in these patients. Complexes between p53 protein and a 70kDa heat shock protein might be necessary for the antigenic presentation of p53 (Davidoff et al., 1992). This putative prerequisite may only be fulfilled in a few pancreatic carcinomas.
In previous analyses (Kalthoff et al., 1993;Kessler et al., 1993) we were able to show specific p53 immunoreactivity in cytospin preparations derived from patients with pancreatic cancer (7/10) and, in addition, in specimens from patients with acute and chronic pancreatitis (9/13). The fact that antibodies against p53 were found in serum samples of patients with non-malignant pancreatic diseases may point to a p53 release and antigen processing with subsequent elicitation of a p53-directed antibody response by necroinflammatory benign diseases. Another hypothesis to explain the occurrence of p53 antibodies in these patients is the presence of as yet undetected malignancies.
We are grateful to Professor Thierry Soussi, Paris, for kindly providing his ELISA sstem and to Professor Wolfgang Deppert, Hamburg, for kindly providing recombinant wt p53 protein We acknowledge the excellent technical assisance of Britta Busing. We thank Alan I AdakI London, for critical reading of the man t. This study was supported by the E.