Toxicokinetics of the phytoestrogen daidzein in female DA/Han rats.

Female DA/Han rats were given the phytoestrogen daidzein, either intravenously (10 mg/kg b.w.) or orally by gavage (10 or 100 mg/kg b.w.). The plasma concentration-time curve determined after i.v. administration of daidzein was fitted to a triexponential model, resulting in a final half-life (gamma-phase) of approximately 4 h. The oral bioavailability of 10 mg daidzein/kg was 9.7%, while that of 100 mg/kg was 2.2%; the higher dose (100 mg/kg) was apparently absorbed to a four- to fivefold lower extent than the smaller dose. The plasma concentration time curves after oral administration of daidzein to female DA/Han rats revealed pronounced interindividual differences and multiple peaks, pointing to extensive enterohepatic circulation and/or protracted absorption from the gastrointestinal tract. As shown in a separate experiment with bile duct-cannulated rats, daidzein (i.p. 10 mg/kg b.w.) is efficiently excreted with bile: glucuronide/sulfate metabolites amounting to approximately 30% of the dose in 8 h. Conjugates were also the main circulating metabolites upon i.v. or gavage administration of daidzein, indicating efficient phase II metabolism in female DA/Han rats. Since only few data have been published on tissue levels of isoflavones, their concentrations were measured in various organs and compared to plasma levels determined at the time the animals were killed, with one exception 32 or 48 h after rats had received a single dose of daidzein (i.v. or per os). As expected, the daidzein concentrations depended upon dose and administration route. Despite notable differences in the absolute amounts of total daidzein (free plus hydrolyzed conjugates), the levels were usually three- to fivefold higher in liver and kidney than in plasma; in most samples of uteri, the concentrations were similar, or up to twofold higher, than the respective plasma levels. These data point to an uptake and storage of isoflavones and metabolites in tissues. Experimental toxicokinetics appear to be a relevant subject that should be integrated into assessments of toxicological data for endocrine modulators.

Toxicokinetics of p-tert-octylphenol in female DA/Han rats after single i.v. and oral application.

Female DA/Han rats were administered p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol], either intravenously (5 mg/kg body wt.) or orally by gavage (50 or 200 mg/kg body wt.). After i.v. administration the blood concentration-time curve of OP was fitted to a tri-exponential model, resulting in a final half-life (gamma-phase) of 36.1 h. This contrasts to much more rapid eliminations previously reported in male Wistar rats. The oral bioavailability of 50 mg/kg OP was 12.3% and of 200 mg/kg 8.4%. The higher dose (200 mg kg) was absorbed slower than the smaller dose, probably due to low solubility of OP in aqueous media. Maximal OP blood levels in female DA/Han rats receiving 50 and 200 mg OP/kg body wt, were 4.5 and 3 times higher than previously reported in male Wistar rats. The blood concentration-time curves after oral administration of OP to female DA/Han rats revealed pronounced interindividual differences, indicating extensive enterohepatic circulation of OP in this rat strain. In contrast to male Wistar rats, after application of high doses of OP to female DA/Han rats the compound was not completely eliminated within 48 h: under these conditions some bioaccumulation might therefore occur. The experimental toxicokinetics of OP appears as a relevant subject to be integrated into extrapolation of toxicological data, from in vitro to in vivo, and into systems of risk assessment of endocrine modulating activity which are currently being developed.

Modulation of prostaglandin H synthase-2 mRNA expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice.

Prostaglandin endoperoxide H synthases (PGHS-1 and PGHS-2) catalyze an intermediate step in the biosynthesis of prostaglandins and thromboxanes. Recently, it was observed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates the expression of PGHS-2 mRNA in different cell lines. The main aim of this study was to examine whether PGHS-2 mRNA expression can be changed by acute TCDD in vivo and, second, we were also interested in whether modulation of PGHS-2 is mediated by the aryl hydrocarbon receptor (AhR) which is known to be involved in the transcriptional control of TCDD-induced phase 1 and phase 2 enzymes. Initially C57BL/6J mice were treated with a single dose of 10,000 ng TCDD/kg and the PGHS-1 and PGHS-2 mRNAs were analyzed in liver, lung, thymus, kidney, and spleen. In all tissues examined the expression of PGHS-1 mRNA was not affected by TCDD. However, TCDD treatment enhanced the PGHS-2 mRNA levels in lung and spleen. No effect of TCDD on PGHS-2 expression was found in liver and kidney. For dose-response studies C57BL/6J and DBA/2J mice were treated for 24 h with various doses of TCDD (1-50,000 ng/kg) and the PGHS-2 mRNA increases were analyzed in lungs and spleens. A significant increase of PGHS-2 mRNA in lungs of C57BL/6J mice was found at a dose of 100 ng TCDD/kg, whereas a nearly 100-fold higher TCDD dose was needed to increase PGHS-2 in DBA/2J mice. A similar dose-dependent induction of PGHS-2 was found in spleens of C57BL/6J mice; however, no significant increase of PGHS-2 was found in spleens of DBA/2 mice. These results indicate an involvement of AhR in TCDD-mediated changes of PGHS-2 expression. This suggestion is supported by studies in AhR-deficient animals which showed that TCDD had no effect on PGHS-2 mRNA. When changes of PGHS-2 mRNA expression are compared with those of CYP1A1 between 4 and 72 h after TCDD, it is noteworthy that TCDD led to a delayed and more transient increase of PGHS-2. These data suggest that the mechanism of modulation of both genes by TCDD may be different.

Deoxynivalenol and Nivalenol Production by Fusarium culmorum Isolates Differing in Aggressiveness Toward Winter Rye.

ABSTRACT A susceptible synthetic winter rye population was inoculated with 42 isolates of Fusarium culmorum, originating from nine European countries and Australia, at two field locations in Germany. Significant (P = 0.01) genetic variation in aggressiveness of isolates of F. culmorum was observed across both field locations. Field samples were used to determine deoxynivalenol (DON), nivalenol (NIV), and ergosterol (ERG) contents. The 42 isolates also were incubated on rye grain in vitro, and DON and NIV contents were analyzed. Thirty-four isolates produced DON, and seven isolates produced NIV at both field locations and in vitro. Mean DON contents ranged from 0.5 to 64.6 mg/kg in grain from field trials and from 0.3 to 376.3 mg/kg in grain incubated in vitro; mean NIV contents ranged from 17.6 to 30.4 mg/kg in grain from field trials and from 0.8 to 381.0 mg/kg in grain incubated in vitro. No correlation was found between the DON content of field-grown grain and grain incubated in vitro. NIV-producing isolates originated from the Netherlands, Germany, Italy, and Australia. More aggressive isolates produced higher mean DON contents in grain in field trials (r = 0.69; P = 0.01). However, DON production rate per unit of fungal biomass, estimated as the DON/ERG ratio at harvest, was not correlated with aggressiveness. Toxin production seemed to be a common feature in F. culmorum. In vitro assays reliably distinguished DON- and NIV-producing types of F. culmorum; however, these assays could not predict production of DON by these isolates in the field.

[Signal improvement of pulmonary venous Doppler flow prifile after intravenous injection of levovist]. / Signalverbesserung des pulmonalvenösen Dopplerflussprofils nach intravenöser Injektion von Levovist.

The analysis of the pulmonary venous flow Doppler pattern can assist in the determination of the severity of mitral regurgitation and, in conjunction with transmitral flow pattern, the assessment of left ventricular diastolic dysfunction. In about one third of the cases, however, transthoracic ultrasonography is not able to record an adequately analyzable pulmonary venous flow pattern. The aim of the study was to examine and compare the effect of the echo-enhancing agent Levovist on the pulsed-wave Doppler flow quality of the transthoracically (TTE) and transesophageally (TEE) recorded pulmonary venous flow. In 26 consecutive patients, a qualitative (score system) and quantitative analysis of the pulmonary venous flow pattern was obtained before and after peripheral venous injection of Levovist at concentrations of 200 mg/ml (low dose) and 400 mg/ml (high dose). The number of measurable studies for the antegrade pulmonary venous flow increased after Levovist from 85% to 96% for TTE and from 96% to 100% for TEE. The retrograde flow as seen by TTE was adequately analyzable in only 45% before and in 73% after injection of Levovist (p < 0.02). Before any contrast enhancement, the retrograde pulmonary venous flow recorded by TEE could be analyzed in 77% of the patients with the percentage increasing to 88% and 92% after administration of a low and high dose of Levovist, respectively (p < 0.05). In particular, the quality score of the retrograde flow was significantly altered by the administration of Levovist (increase from 1.8 +/- 1.0 to 2.6 +/- 1.1 (low dose Levovist), p < 0.05 and to 2.7 +/- 1.3 (high dose Levovist). p < 0.05). The pulsed-wave Doppler evaluation by TTE without Levovist underestimated the velocities of the antegrade and retrograde pulmonary venous flow After administration of Levovist, the recorded values are comparable to those obtained by TEE. An analogous pattern is encountered when quantifying the duration of the retrograde flow component. Thus, the peripheral venous injection of Levovist leads to an improved quality of the pulmonary venous flow Doppler signal recorded by TTE. Qualitatively and quantitatively the values recorded by TTE after administration of Levovist are comparable to those of the TEE technique without an echo-enhancing agent.

An in vitro model of the urinary bladder: cultured porcine urinary bladder epithelial cells.

Epithelial cells from urinary bladders of pigs were isolated and cultured under serum-free conditions. For these cells it was previously shown that they developed morphologic polarity resembling the epithelium in vivo. Lactate dehydrogenase release was low, chromosome set and activities of marker enzymes (alkaline phosphatase, acid phosphatase, g-glutamyltranspeptidase) were stable over a period of 4 wk. In this study, metabolic competence was evaluated by measuring activities of phase I and phase II enzymes. Activity of prostaglandin H-synthase was expressed in freshly isolated cells as well as in cultured cells, as were activities of the conjugating enzymes glutathione transferase, UDP-glucuronyltransferase and N-acetyltransferase. Cytochrome P4501A1 activity in freshly isolated cells amounted to 10-15% of the respective activity in the porcine liver, this activity was not detectable in cultured cells. No activity was seen in cultured cells after induction with methylcholanthrene and benz[a]anthracene. This cell culture system was used to detect genotoxic effects of substances suspected to induce bladder cancer by measuring the induction of sister chromatid exchanges (SCE). The aromatic amines 4-aminobiphenyl and 2-aminofluorene induced a concentration dependent increase of SCEs at non-cytotoxic concentrations. These results imply that urinary bladder epithelial cells are capable to perform metabolic activation which is required to generate genotoxic effects of aromatic amines. Therefore, this new cell culture system, representing the urinary bladder epithelium, is an effective tool in in vitro toxicology to investigate adverse effects of compounds, regarded or suspected to induce toxic effects in the bladder.

Improvement of pulmonary venous flow Doppler signal after intravenous injection of Levovist.

The analysis of the pulmonary venous flow Doppler pattern can assist in the determination of the severity of mitral regurgitation and, in conjunction with transmitral flow pattern, the assessment of left ventricular diastolic dysfunction. In approximately one third of the cases, however, transthoracic ultrasonography is not able to record an adequately analyzable pulmonary venous flow pattern. The aim of the study was to examine and compare the effect of the echo-enhancing agent Levovist on the pulsed-wave Doppler flow quality of the transthoracic (TTE) and transesophageal (TEE) recorded pulmonary venous flow. In 26 consecutive patients a qualitative (score system) and quantitative analysis of pulmonary venous flow pattern was obtained before and after peripheral venous injection of Levovist at concentrations of 200 mg/ml (low dose) and 400 mg/ml (high dose). The amount of measurable studies for the antegrade pulmonary venous flow increased after the injection of Levovist from 85% to 96% for TTE and from 96% to 100% for TEE. The retrograde flow as seen by TTE was adequately analyzable in only 46% before and in 73% after the injection of Levovist (p < 0.02). Before any contrast enhancement the retrograde pulmonary venous flow recorded by TEE could be analyzed in 77% of the patients, with the percentage increasing to 88% and 92% after the administration of a low and high dose of Levovist, respectively (p < 0.05). In particular, the quality score of the retrograde flow was significantly altered by the administration of Levovist (increase from 1.8 +/- 1.0 to 2.6 +/- 1.1 [low dose Levovist], p < 0.05 and to 2.7 +/- 1.3 [high dose Levovist], p < 0.05). The pulsed-wave Doppler interrogation by TTE without Levovist underestimated the velocities of the antegrade and retrograde pulmonary venous flow. After Levovist was administered, the recorded values were comparable to those obtained by TEE. An analogous pattern is encountered when quantifying the duration of the retrograde flow component. Thus the peripheral venous injection of Levovist leads to an improved quality of the pulmonary venous flow Doppler signal recorded by TTE. On qualitative and quantitative evaluation the values recorded by TTE after administration of Levovist are comparable to those of the TEE technique without an echo-enhancing agent.

Drug metabolizing enzyme activities in porcine urinary bladder epithelial cell cultures (PUBEC).

Drug metabolizing enzyme activities have been determined in cultured porcine urinary bladder epithelial cells (PUBEC) in order to evaluate this system as an in vitro model for studies of urinary bladder carcinogens. Activities of several phase I and II enzymes were measured in cells cultured for various periods and compared with the activities determined in freshly isolated PUBEC. Prostaglandin H synthase mediated production of prostaglandin E2 was found both in freshly isolated and in cultured PUBEC, whereas cytochrome P450 1A1-associated EROD activity was only detectable in freshly isolated bladder cells. The latter activity was not inducible by benz(a)anthracene or 3-methylcholanthrene in PUBEC cultures. N-acetyltransferase (NAT) activity measured with p-aminobenzoic acid, a diagnostic substrate for human NAT-1, was stable and even higher during the culture period compared to freshly isolated cells. In contrast, isoniazid (a substrate for NAT-2) was not acetylated either in fresh or cultured PUBEC. Glutathione S-transferases activity determined with 1-chloro-2,4-dinitrobenzene decreased gradually to 50% after 1 week and to 20% after 4 weeks in culture compared to fresh cells. A similar decline was also observed for UDP-glucuronyltransferase activities measured with 1-naphthol. In accordance with the reported lack of sulfotransferases in pigs, no sulfation of 1-naphthol or 2-naphthylamine was detected in PUBEC. Our results show that cultured porcine urinary bladder epithelial cells maintain several enzyme activities required for the biotransformation of xenobiotics. In future investigations on the mechanism of action of bladder carcinogens PUBEC cultures may thus provide a useful in vitro model for this target tissue.

Induction of mono-oxygenase activity in prostaglandin H synthase-competent ovine seminal vesicle cell cultures by 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Cell cultures derived from ovine seminal vesicles (OSV cells) express high levels of prostaglandin H synthase (PHS) and have been found to be a suitablein vitro model for studies on PHS-mediated bioactivation of certain xenobiotics The extrahepatic tissue of origin apparently lacks constitutively expressed mono-oxygenase (MFO) activity such as cytochrome P-450 (CYP1A1)-dependent ethoxyresorufin-O-deethylase (EROD). However, treatment of OSV cell cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.001-10 nm) resulted in a dose-dependent induction of CYP1A1-associated EROD activity. Subconfluent OSV cells exposed to 100 pm TCDD for various lengths of time express the highest EROD activity after 48 hr of treatment. Benz[a]anthracene also induced EROD activity in OSV cells, but, in accordance with its low affinity for the Ah receptor at much higher concentrations (1-10 mum) than TCDD. In line with the observed functional response (EROD induction) the presence of Ah receptor was confirmed by biochemical analysis: on incubation with [(3)H]TCDD 5 fmol ligand/mg nuclear protein were specifically bound to Ah receptor after incubation (2 hr) of OSV cells with [(3)H] TCDD. TCDD was not cytotoxic for OSV cells up to 10 nm as judged by growth curves; rather, their growth was moderately stimulated by TCDD, significantly at 1 nm (a concentration which also induced EROD activity). Despite a clear dose-related response of OSV cells to known inducers of CYP1A1, the induced EROD activity levels (0.4-0.7pmol/minx 10(6)cells) are very low compared with values reported for hepatic cells in culture and lower than EROD activity induced in colon tumour cells. Thus, and since OSV cells do not constitutively express appreciable MFO activity, it is suggested that only PHS-mediated oxidation reactions contribute to the bioactivation of xenobiotics in this model. In conclusion, although OSV cells clearly respond to Ah receptor ligands by EROD induction, they are of limited value for bioassay of TCDD and related compounds in environmental samples. However, PHS-competent OSV cells are an interesting model for further studies of TCDD-related effects on PHS activity/expression, because of the functional Ah receptor.

[Intraperitoneal fetal small intestine transplantation in allogeneic rat strains]. / Intraperitoneale fetale Dünndarmtransplantation bei allogenen Rattenstämmen.

We demonstrated the possibility of a successful transplant of foetal rat small intestine into the peritoneal cavity of adult rats. Several combinations of rat strains with defined genetic differences were chosen. On the whole, in more than 66 percent of the performed transplantations a successful adequate growth of the transplants was noted. Oral application of Cyclosporin A (20 mg/kg/d) as immunosuppressive to prevent allograft rejection was necessary only in the combination across major genetic barriers. The preoperative insertion of a 7-0 non-absorbable thread into the lumen of the transplant helped to develop a tubular segment of small intestine which can be anastomosed to the intestinal continuity of the host in a second stage operation.

[Occurrence of various enzymes in synovial fluid of cattle and swine]. / Zum Vorkommen einiger Enzyme in der Gelenkflüssigkeit von Rind und Schwein.

The activities of lactate dehydrogenase (LDH), fructose-1.6-diphosphate aldolase (ALD), aspartate aminotransferase (AspAT), alkaline phosphatase (AP), and N-acetyl-beta-glucosaminidase (NAG) were determined on the basis of 75 synovia samples taken from the tarsal joints (Art. talocruralis) of 41 cattle for slaughter of different sexes and aged between one and 13 years as well as on the basis of 56 synovia samples taken from the knee joints (Art. femoropatellaris), tarsal joints (Art. talocruralis), and carpal joints (Art. intercarpicus) of 20 fattening pigs. Both the general condition and cell content of synovial fluid in clinically intact joints are described. The activities of ALD and AspAT (less than 15 IU/l), LDH (less than 200 IU/l), and NAG (less than 6,000 IU/l) in synovial fluid of cattle were much lower than those in blood serum of the same species. They were normally distributed. AP activity (less than 150 IU/l) in synovial fluid, however, was higher by several factors as compared to activity in blblished. In swine synovial AspAT and AP activities were just as high as in blood serum, while LDH activities were higher by 1.5 times. Major NAG activity was observed, as well. All enzyme activities were characterised by normal distribution. All five LDH isoenzymes but only one AP isoenzyme were established. The above data were compared with findings reported by other authors, and the comparison showed these results as being characteristic of synovial enzyme activities in clinically intact joints of the two species under review.

[Lactate dehydrogenase (LDH) and isoenzymes in pig liver during postnatal development]. / Das Verhalten der Laktatdehydrogenase und ihrer Isoenzyme in der Ferkelleber während der postnatalen Entwicklung.

1. LDH-activity in the liver of newborn piglets is 4 times that of adult pig sliver and remains relatively constant at this level during the first 3 weeks of life. 2. 90% of LDH is localized in the cytosolic compartment of the liver, independent of age of pigs. 3. In the liver of newborn piglets the isoenzymes LDH-3 and LDH-4 includes the main part of LDH-activity. The typical distribution of LDH isoenzymes in the liver of adult pigs (LDH-3 greater than LDH-2 greater than LDH-1 greater than LDH-4 greater than LDH-5) is already reached in 6-day-old piglets. Afterwards there follows only a decrease of LDH-1. Therefore, during postnatal development a directed shift from M- to H-type of isoenzyme distribution is observed.

[Effect of the type of extraction solution and length of storage on the distribution of lactate dehydrogenase isoenzymes in piglet liver homogenate]. / Der Einfluss der Art des Extraktionsmittels und der Dauer der Aufbewahrung auf die Verteilung der LDH-Isoenzyme im Homogenat der Ferkelleber.

Type of extraction solution applied to preparation of homogenate of piglet liver is of negligible importance, if immediately follows electrophoretic separation of tissue material. --Storage of liver homogenates in frozen state over 30 days involves insignificant changes of distribution of LD isoenzymes in 0.25 M sucrose, distinct changes in 0.1 M Tris-buffer, pH 7,5, and important ones in mercaptoaethanol-containing extraction solution, applied to preparation of tissue homogenates.