Quantification of the xenoestrogens 4-tert.-octylphenol and bisphenol A in water and in fish tissue based on microwave assisted extraction, solid-phase extraction and liquid chromatography-mass spectrometry.

Extraction methods were developed for quantification of the xenoestrogens 4-tert.-octylphenol (tOP) and bisphenol A (BPA) in water and in liver and muscle tissue from the rainbow trout (Oncorhynchus mykiss). The extraction of tOP and BPA from tissue samples was carried out using microwave-assisted solvent extraction (MASE) followed by solid-phase extraction (SPE). Water samples were extracted using only SPE. For the quantification of tOP and BPA, liquid chromatography mass spectrometry (LC-MS) equipped with an atmospheric pressure chemical ionisation interface (APCI) was applied. The combined methods for tissue extraction allow the use of small sample amounts of liver or muscle (typically 1 g), low volumes of solvent (20 ml), and short extraction times (25 min). Limits of quantification of tOP in tissue samples were found to be approximately 10 ng/g in muscle and 50 ng/g in liver (both based on 1 g of fresh tissue). The corresponding values for BPA were approximately 50 ng/g in both muscle and liver tissue. In water, the limit of quantification for tOP and BPA was approximately 0.1 microg/l (based on 100 ml sample size).

Uptake, metabolism and excretion of bisphenol A in the rainbow trout (Oncorhynchus mykiss).

The uptake, metabolism and excretion of the oestrogenic chemical bisphenol A (BPA) were studied in juvenile rainbow trout (Oncorhynchus mykiss). BPA was detectable in plasma, liver and muscle after 2 h of water exposure at 0.44 microM (100 microg BPA/l), and a steady state was reached within 12-24 h. The concentration of the glucuronidated degradation product in the plasma was about twice that of the parent compound. A plasma half life of BPA was calculated as 3.75 h following injection of the compound. The vitellogenin synthesis was measured in response to the BPA treatment, and a lag period of 5 and 7 days between injection of the compound and a significant vitellogenin response was observed for females and males, respectively. At the time of the vitellogenin response no BPA could be detected in the liver tissue from either male or female fish. These results indicate that fish briefly exposed to elevated levels of oestrogenic chemicals might develop a response several days later.

In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncorhynchus mykiss).

The in vivo estrogenic activity of the two branched alkylphenols, tert-octylphenol and technical nonylphenol, and the two linear isoforms, n-octylphenol and n-nonylphenol, was compared. The compounds were administered to juvenile rainbow trout (Oncorhynchus mykiss) by either intraperitoneal injection or water exposure and their estrogenic potential was evaluated by ELISA measurements of induced plasma vitellogenin. Intraperitoneal injections (50 mg/kg) of the two branched alkylphenols resulted in a significant vitellogenic response after 12 days whereas no significant induction was seen with the two linear isomers. Water exposure for 9 days to a nominal concentration of 150 micrograms/l of the alkylphenols elicited the same response pattern as seen for the injection experiment. Furthermore, in the present vitellogenin assay tert-octylphenol was giving a higher estrogenic response compared to technical nonylphenol using either of the two exposure routes.

Induction and identification of cadmium-, zinc- and copper-metallothioneins in the shore crab Carcinus maenas (L.).

Shore crabs Carcinus maenas were injected with either Cd, Cu or Zn to determine whether different metals could induce specific metallothionein (MT) isoforms in the midgut gland. Furthermore, the relative ability of the three metals to induce MT was quantified. Accumulation of the three metals in the midgut gland caused variable and in the case of Cd and Zn significant increases in MT levels. The increase in MT levels (pmol g-1 midgut gland) per nmol of metal accumulated was determined as 90, 60 and 4 pmol for Cd, Zn, and Cu respectively. The MT isoforms were purified using a combination of acetone precipitation, FPLC and reverse phase HPLC. In contrast to Cd and Zn induced MTs, the Cu induced MT was highly susceptible to oxidation during purification. The induced MT isoforms were characterized by N-terminal amino acid sequencing and mass-spectrometry. All three metals induced the same identical isoform MTIa.

Arsenic biotransformation by the brown macroalga, Fucus serratus.

The brown alga Fucus serratus was maintained in aquaria with added arsenate (0, 20, 50, and 100 microg As/L, four individuals per treatment) for up to 19 weeks. Biotransformation of arsenic by Fucus was monitored by high-performance liquid chromatography/inductively coupled plasma mass spectrometry and liquid chromatography/electrospray mass spectrometry analysis of aqueous extracts of algal frond tips removed periodically throughout the experiment. Major arsenic species monitored were arsenate, arsenite, methylarsonate, dimethylarsinate, and the four arsenosugars 1 to 4 found naturally in Fucus. Algae accumulated arsenate readily and transformed it into several arsenic compounds depending on the exposure concentration. At 100 microg As/L, the major metabolite was arsenite with smaller quantities of methylarsonate and dimethylarsinate, but only traces of arsenosugars were formed. In contrast, the 20-microg-As/L group accumulated only small quantities of arsenite and methylarsonate, while dimethylarsinate and arsenosugars were major arsenic metabolites. At 50 microg As/L exposure, algae had significant quantities of all arsenic metabolites monitored. Arsenate was toxic to the algae at 100 microg As/L but had no obvious detrimental effect at 20 microg As/L. The data are consistent with a process of arsenate detoxification by reduction and alkylation; at higher exposures, however, the alkylation processes become saturated, leading to an accumulation of arsenite and subsequent toxicity.

[Phlegmonous gastritis in pregnancy--a case with fatal course]. / Flegmonøs gastritis i graviditeten--et dødeligt forløbende tilfaelde.

A case of maternal death and still birth in the 35th week of pregnancy is described where the cause of death was the very rarely diagnosed phlegmonous gastritis. The phlegmonous gastritis is characterised by dangerous, fulminating pyogenic bacterial infection of the gastric wall with alpha-haemolytical streptococci. The entity seems to have been well-known earlier, but has only been reported sparsely in contemporary medical literature. It cannot be ruled out that the disease may have been misdiagnosed on several occasions in recent decades because of symptomatic treatment. In presenting the case, the authors seek to attract attention to this disease--that according to the contemporary medical literature seems mostly to be induced by modern treatment with antacids and after gastroscopy--because it can be effectively treated with modern antibiotics, possibly in combination with gastrectomy.

The glycolytic enzyme activity of the human cervix uteri.

The activity of the following enzymes was studied in normal, precancerous, and malignant biopsies from the human cervix uteri: hexokinase (HK), phosphofructokinase (PFK), pyruvate-kinase (PK), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G-6-PDH). In precancerous conditions, i.e., dysplasia and carcinoma in situ without any signs of invasive carcinoma, only PK showed moderate but significant activity increases. A rise in enzyme activity in biopsies histologically classified as carcinoma in situ was found to signal the presence of invasive carcinoma in other parts of the cervix. In invasive carcinomas of the cervix, all the enzymes studied showed a two- to four-fold increase (p less than 0.01) as compared to the normal cervix. The present study failed to reveal significant differences between enzyme activities in biopsies from patients in Stage I, II, and III; no correlation could be established between enzyme activity and prognosis.

Enzymatic studies of glycogen metabolism in nonmalignant and malignant biopsies from the human uterine cervix.

The glycogen metabolism of the human uterine cervix has been investigated in tissue specimens from 147 women with gynaecological diseases: 64 with cervical carcinomas, 6 with carcinoma in situ and 77 with nonmalignant cervical diseases. The glycogen content of the normal uterine cervix was found to be fairly constant and apparently not regulated by steroid hormones. The low content of glycogen in malgnant cervical tumours was confirmed by the present investigation. The percent investigation also showed that this abnormality was not only characteristic of invasive carcinoma but also of carcinoma in situ. A particularly low glycogen content was found in tissue samples from patients with relapse. Concerning the enzyme involved in glycogen metabolism, a significantly high activity of glycogen synthetase was found in malignant cervical biopsies, while the activity of glycogen phosphorylase did not differ significantly when comparing normal to malignant tissue.

Liquid chromatography electrospray mass spectrometry with variable fragmentor voltages gives simultaneous elemental and molecular detection of arsenic compounds.

A single quadrupole high performance liquid chromatography electrospray mass spectrometry system with a variable fragmentor voltage facility was used in the positive ion mode for simultaneous recording of elemental and molecular mass spectral data for arsenic compounds. The method was applicable to the seven organoarsenic compounds tested: four arsenic-containing carbohydrates (arsenosugars), a quaternary arsonium compound (arsenobetaine), dimethylarsinic acid, and dimethylarsinoylacetic acid. It was not suitable for the two inorganic arsenic species arsenite and arsenate. In the case of arsenosugars, qualifying ion data for a characteristic common fragment (m/z 237) was also simultaneously obtained. The method was used to identify and quantify the major arsenosugars in crude extracts of two brown algae.

Estriol concentrations in urine and serum in patients with various intestinal diseases.

Pregnant women with ulcerative colitis shows frequently low estriol values in both serum and urine. 18 patients with ulcerative colitis, 4 patien;s with mb. Crohn, and 3 patients with a by-pass operation were examined. Low estriol concentrations were seen only in the patients with ulcerative colitis especially in patients, where an operation has been performed. In one patient with severe diarrhea the estriol concentration in serum was low until the intestinal function normalised and the estriol concentration went up exactly when the diarrhea stopped. However, no unequivocal connection between low estriol concentrations and diarrhea could be demonstrated.

The preservatives ethyl-, propyl- and butylparaben are oestrogenic in an in vivo fish assay.

The widely used phenolic preservatives ethylparaben, propylparaben, butylparaben and their common metabolite p-hydroxybenzoic acid were tested for their ability to evoke an oestrogenic response in vivo. Yolk protein induction in sexually immature rainbow trout was used as an oestrogen-specific endpoint after repeated injections of the compounds. All tested parabens were oestrogenic in doses between 100 and 300 mg/kg, while the metabolite showed no activity. Ethylparaben was found to be approximately sixty times weaker than propyl- and butylparaben which had oestrogenic potencies comparable to those previously found for bisphenol A.

Metabolism of bisphenol A in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) in relation to estrogenic response.

The kinetics of bisphenol A (BPA) were investigated in zebrafish (Danio rerio) exposed to 100 microg BPA/l. BPA uptake was measured during a 7-day period followed by an elimination phase of similar duration. After 2, 6, 12, 24, 48, 72, 120 and 168 h of uptake/elimination, fish were analysed for their content of BPA, bisphenol A glucuronic acid (BPAGA) and bisphenol A sulfate (BPAS). Within the first 24 h steady state levels of BPA, BPAGA and BPAS were reached and the total body concentrations were calculated to be 569, 12,600 and 39.9 ng/g fish, respectively. Elimination rates of the three compounds in zebrafish were estimated by fitting the data to a compartment model. An initial rapid elimination phase was observed for BPA and BPAS with total body half lives (T(1/2)) of <1.1 h and 30 min, followed by a slower second elimination phase with T(1/2) values of 139 and 71 h, respectively. Excretion of BPAGA occurred from a single compartment with a T(1/2) of 35 h. The steady state concentration of BPA and its metabolites were investigated in rainbow trout (Oncorhynchus mykiss) exposed to 100 microg BPA/l. The toxicokinetic parameters from zebrafish and rainbow trout were compared; including previously published data on the rainbow trout. The data indicate that the smaller estrogenic sensitivity observed for the zebrafish may be caused by a more rapid metabolism of BPA in the zebrafish liver.

Primary structures of decapod crustacean metallothioneins with special emphasis on freshwater and semi-terrestrial species.

Cadmium injections induced only a single form of metallothionein (MT) in the midgut gland of Potamon potamios, whereas the same treatment induced two isoforms in Astacus astacus. The only difference between the two latter isoforms was that one had an extra N-terminal methionine residue. MT from P. potamios showed structural differences from other decapod crustacean MTs. It contained a Gly-Thr motif at positions 8 and 8a, which had previously been found only in certain vertebrate and molluscan MTs. Furthermore P. potamios MT contained two to three times as many glutamic acid residues as normally found in decapod crustacean MT. The primary structure of MT from the freshwater crayfish A. astacus showed a high degree of sequence identity with MT from other decapod crustaceans, especially the marine astacidean Homarus americanus, although two valine residues were unexpectedly found at positions 8 and 21, where lysine residues are normally found.

Determination by photoaffinity labelling of the hydrophobic part of the binding site for acyl-CoA esters on acyl-CoA-binding protein from bovine liver.

Acyl-CoA esters containing the photoreactive acids 12-(4'-azido-2'-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4'-azido-2'-nitro-[3'-5'-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4'-Azido-2'-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4'-azido-2'-nitro[3',5'-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond.

Purification and characterization of a cadmium-induced metallothionein from the shore crab Carcinus maenas (L.).

Two metallothionein variants were purified from the midgut gland of crabs (Carcinus maenas) exposed to a high cadmium concentration (2 p.p.m.). One of the variants was purified from crabs exposed to a low cadmium concentration (0.5 p.p.m.). The purification method involved acetone precipitation, gel filtration and reversed-phase h.p.l.c. The complete amino acid sequences of both variants have been elucidated by m.s. and automated sequence analysis on S-methylated proteins or fragments produced by cleavage of the S-methylated proteins with Staphylococcus aureus proteinase. The two variants from crabs exposed to the high cadmium concentration differed only by a single residue of methionine at the N-terminus. The single variant isolated from crabs exposed to the low cadmium concentration was the one without the N-terminal methionine, indicating that high cadmium concentrations either inhibit the processing enzymes and/or that the processing enzymes cannot keep pace with the increased metallothionein synthesis when cadmium availability is high. Cadmium-induced metallothionein from C. maenas shows a high degree of structural similarity to metallothioneins from the decapod crustaceans Scylla serrata and Homarus americanus.