Christian Schlatter

The occurrence of ochratoxin A in coffee.

Ochratoxin A (OA) is a nephrotoxic and nephrocarcinogenic mycotoxin which is predominantly produced by the two ubiquitous fungal genera, Aspergillus and Penicillium. OA is found in foodstuffs, predominantly in cereals but also in coffee beans. Inconsistent results have been published regarding the influence of roasting on the OA content in roasted beans and the transfer into the coffee brew. In the present study an HPLC method was used for the detection of OA in green and roasted coffee beans as well as in the coffee brew. For qualitative confirmation and quantification of low OA levels in roasted coffee beans and coffee brew an additional clean-up step by immunoaffinity column was applied before HPLC analysis. In green coffee beans OA was detected in 13 out of 25 commercial samples analysed (detection limit, 0.5 micrograms OA/kg). Roasting (250 degrees C, 150 sec) of naturally contaminated green beans or beans inoculated with A. ochraceus resulted only in a small reduction in the OA level. OA was also found to be eluted into the brew. Of 40 coffee brews prepared from commercially available samples OA was detected in 18 brews by HPLC and/or additional immunoaffinity column clean-up in the range of 0.4 to 7.8 micrograms OA/kg equivalent ground coffee. Our preliminary results suggest, therefore, that regular coffee consumption may contribute to exposure of humans to OA.

Toxicity of Microcystis aeruginosa peptide toxin to yearling rainbow trout (Oncorhynchus mykiss)

Abstract The effects of the blue-green algae (cyanobacterial) toxin microcystin-LR on yearling rainbow trout were studied in a series of intraperitoneal injection tests, gavage trials and exposures to waterborne algae. Concentrations of the microcystin-producing algae Microcystis aeruginosa known to occur during algae blooms (8–16 mg freeze-dried algae/L, i.e. approximately 1–2 × 1011 cells/L) were shown to be non-toxic to trout when present in aquarium water. On the other hand, trout died within 96 h when gavaged with an amount of algae equivalent to that which passed through the gills within 18 h in the aqueous exposure tests (1440 mg freeze-dried algae/kg body weight, i.e. 6600 μg microcystin/kg body weight). Oral uptake of approximately one tenth of this dose (110 mg freeze-dried algae/kg body weight, i.e. 550 μg microcystin/kg body weight) proved to be non-toxic in single gavaging studies, but toxic within 96 h when orally administered 8 times at 12-h intervals. This study shows that the main uptake route of microcystin in trout is the gastrointestinal tract and that toxicity is manifested as massive hepatic necrosis. These results indicate that massive fish deaths reported at cyanobacterial bloom sites can be explained by the ingestion of toxic blue-green algae.

Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo

It was the aim of this investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or without pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [14C]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The limit of detection, CBI less than 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [14C]- and [3H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside or base adducts are expected. The respective limits of detection were at 0.07 and 0.04 CBI units for the 14C and 3H labels, respectively. The experiments with [14C]- and [3H]DEHA, labeled in the alcohol moiety and administered to mice, revealed a minute radioactivity of less than 50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which had been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for 14C and 0.01 to 0.12 for 3H resulted. Determination of the level of 14CO2 expiration revealed a linear correlation with the specific activity of DNA. Experiments with 2-ethyl[1-14C]hexanol performed with both rats and mice allowed the conclusion that most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01.(ABSTRACT TRUNCATED AT 400 WORDS)

Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, dieldrin and other inducers.

Abstract Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat liver nuclei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo- p -dioxin (TCDD, 0.01 mg/kg) with an induction to 500 and 300 per cent for nuclei and microsomes, respectively, after 2 days, and to 400 per cent for both after 12 days. Phenobarbital (PB) was given continuously in the drinking water (1 g/l) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. the nuclear activity was only slightly induced to a Constant level of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daily i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down to control values after 12 days. Other inducers tested were benz[a]anthracene (BA), hexachlorobenzene (HCB) and 1,1,1-trichloro-2,2- bis ( p -chlorophenyl)ethane (DDT). The induction pattern with BA was similar to that of MC, a model compound for the group of cytochrome P448 inducers. the induction by HCB and DDT resembled that by PB, a typical cytochrome P450 inducer.

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat, mouse and pig.

[14C]Aflatoxin B1 (AFB1) was isolated from cultures of Aspergillus parasiticus grown on [1-14C]sodium acetate. Covalent binding of AFB1 to liver DNA of rat and mouse was determined 6-8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a Covalent Binding Index (CBI), (micromol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors. The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable. Aflatoxin M1 (AFM1) is a metabolite found in the milk of cows that have been fed AFB1-contaminated diet. [14C]AFM1 was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM1 must also be regarded as a strong hepatocarcinogen. It is concluded that AFB1 contaminations should be avoided in dairy feed.

Covalent binding of ethinylestradiol and estrone to rat liver DNA in vivo.

The covalent binding of [6,7-3H] ethinylestradiol (EE) and [6,7-3H] estrone (E) to liver DNA of 200 g female rats was measured 8 h after the administration of 80 microgram (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressed as binding to DNA/dose, in units of mumol hormone/mol DNA phosphate/mmole hormone/kg body wt. It is the same order of magnitude as for benzene and about 10 000 times below the binding of typical liver carcinogens, such as aflatoxin B1 or N,N-dimethylnitrosamine.

Esterases in the zebra mussel Dreissena polymorpha: activities, inhibition, and binding to organophosphates

Cholinesterase activities of Dreissena polymorpha were measured calorimetrically. In homog- enates of whole control mussel, activities of 125 f29 pmol min- kg- were found (n=6). Neither after exposure of Dreissena to organophosphates (thiometon, disulfoton, demeton-S- methyl) nor after addition of demeton-S-methyl (the activated oxygen analogue of thiome- ton) in vitro was the measured mussel esterase activity inhibited. Esterases of rat, mouse and human tissue showed a 9CL100% inhibition. Radiolabelling of the active serine site of es- terases in muscle homogenates with 3H-diisopropylfluorophosphate and subsequent separa- tion on polyacrylamide gels revealed similarities as well as differences between rat and mussel esterases. Coomassie-stained muscle proteins of Dreissena showed a different distribution pattern than those of rat. Proteins of rat as well as proteins of mussel with molecular weights between 66 and 97 kDa showed best labelling (highest radioactivity). Proteins with molecular weights greater than 97 kDa were not labelled. Additionally, in Dreissena but not in rat, proteins of around 45 kDa were labelled. The results indicate that the esteratic enzymes in Dreissena were labelled but not inhibited by organophosphates.

Cadmium in the Invertebrate Fauna of an Unpolluted Forest in Switzerland

The goal of this investigation was to get a general idea of the distribution of cadmium concentrations in the invertebrate fauna of a woodland area without discernable cadmium pollution from anthropogenic sources. About 1000 individual animals representing over 50 different invertebrate species were sampled. The analysis of whole body cadmium concentrations by graphite-furnace atomic absorption spectrometry revealed an extremely wide range amongst animals found in the same area. Geometric mean concentrations ranged from 5 ng/g dry weight for a June bug to more than 5000 ng/g in snails, earthworms and spiders. Intraspecies variability by itself differed also from species to species: For some the calculated concentration range encompassing 95% of the animals did not span more than a factor of two, for others the concentrations varied by more than a factor of 100.

Biogene Amine in Lebensmitteln: Zur Wirkung von Histamin, Tyramin und Phenylethylamin auf den Menschen

SummaryThe effect of 25 mg histamine, 25 mg tyramine and 5 mg phenylethylamine resp. in apple juice on 27 healthy volunteers was studied using a randomized placebo-controlled double-blind procedure. No statistically significant effect was found with histamine and tyramine, but phenylethylamine produced symptoms like headache, dizziness and discomfort in some volunteers. In a second experiment the effect of four different wines (2 dl) containing naturally several biogenic amines in various amounts (histamine n.d. - 21 ppm; tyramine 1-23 ppm; phenylethylamine n.d. - 6 ppm; putrescine 2-55 ppm) on 20 volunteers was recorded. The percentage of volunteers experiencing symptoms was of the same order of magnitude as in the first experiment. No correlation was found to exist in this second experiment between the occurrence of symptoms and the concentration of biogenic amines in the wine samples.ZusammenfassungDie Effekte von 25 mg Histamin, 25 mg Tyramin and 5 mg Phenylethylamin, verabreicht in Apfelsaft, bei 27 gesunden Probanden wurden in einer randomisierten Placebo-kontrollierten Doppelblind-Prozedur studiert. Keine statistisch signifikanten Effekte wurden mit Histamin and Tyramin registriert, dagegen verursachte Phenylethylamin bei einigen Probanden Symptome wie Kopfschmerzen, Schwindel and Übelkeit. In einem zweiten Experiment wurden die Effekte von vier verschiedenen Weinen (2 dl), die natürlicherweise verschiedene biogene Amine in unterschiedlichen Mengen (Histamine n. n. -21 ppm; Tyramin 1–23 ppm; Phenylethylamin n. n. -6 ppm; Putrescin 2–55 ppm) enthielten, bei 20 Probanden registriert. Der Prozentsatz von Probanden mit Symptomen war in der gleichen Größenordnung wie beim ersten Experiment. Zwischen dem Auftreten von Symptomen and der Konzentration an biogenen Aminen in den verabreichten Wein-Proban wurde keine Korrelation gefunden.

Gehaltsbestimmung von Agaritin im Zuchtchampignon (Agaricus bisporus) mittels Hochleistungsflüssigchromatographie (HPLC)

SummaryA procedure is described for the determination of agaritine in the commercial mushroomAgaricus bisporus by high performance liquid chromatography (HPLC). Agaritine was extracted from the mushroom sample with methanol and the filtered extract diluted with phosphate buffer. An aliquot of this solution was used directly for the HPLC-separation on a cation exchange column (Partisil SCX) with 0.5 mM phosphate buffer (pH 1.8) as mobile phase and u.v. monitoring at 237 nm. The agaritine content in fresh mushrooms was found to be in the range of 94–629 mg/kg fresh weight. Canned mushrooms contained 1–55 mg/kg drained weight with 3–103 mg/l in the liquid. The highest agaritine values were found in dried commercial mushrooms amounting to 2,110–6,905 mg/kg.ZusammenfassungEs wird eine analytische HPLC-Methode zur Bestimmung von Agaritin, einem im Zuchtchampignon (Agaricus bisporus) natürlich vorkommenden Inhaltsstoff beschrieben. Agaritin wurde mit Methanol aus der gefriergetrockneten, homogenisierten Pilzprobe extrahiert und der filtrierte Extrakt mit Phosphat-Puffer verdünnt. Ein Aliquot dieser Lösung wurde direkt verwendet für die HPLC-Auftrennung auf einer Kationenaustauscher-Säule (Partisil SCX) mit Phosphat-Puffer 0,5 mMol/l pH 1,8) als mobile Phase und UV-Detektion bei 237 nm. Die Agaritin-Gehalte in frischen Zuchtchampignons waren im Bereich 94–629 mg/kg Frischgewicht. Abgetropfte Dosenpilze enthielten lediglich 1–55 mg/kg, während im Pilzsaft 3–103 mg/l nachgewiesen wurde. Die höchsten Agaritin-Gehalte (2110–6906 mg/kg) wurden in Trockenpilzen aus dem Handel gefunden.