Rolf Bass

Transfer of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the mouse embryo and fetus

(1) Following the administration of the highly toxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to pregnant mice, exceedingly low concentrations of this substance were found in the embryo and fetus between gestational days 11 and 18 (between 0.04 and 0.14% of the maternal dose/g tissue). (2) Tissue levels of tCDD in embryos on days 9 and 10 exceeded those of later gestational stages. (3) TCDD levels in fetal livers were 2--4 times higher than those in other fetal organs. (4) Maternal liver contained the highest concentrations of TCDD (4 -10% of the dose/g). A comparison of the results obtained following oral, s.c. and i.p. applications indicated that there was no major first pass effect involved following oral administration. (5) Placenta and other extrahepatic maternal organs exhibited TCDD levels, which were approximately 1, embryos and features 2 orders of magnitude lower than maternal liver concentrations. (6) Good correlation was found between the doses applied (5, 12.5 and 25 microgram/kg) and the tissue levels observed. (7) A single dose of 25 microgram/kg given on day 10 or 5 doses of 5 microgram/kg, 1/day from days 7-11, resulted in higher levels of TCDD in embryos on day 13 than did a singly dose of 25 microgram/kg on day 7; cleft palate was induced at different rates by these treatment schedules.

Mammalian mitochondrial ribosomes: Characterization of ribosomal proteins by two‐dimensional gel electrophoresis

Ribosomes isolated from mammalian mitochondria were found to sediment at about 55s [ 1,2] . Investigations on their function as well as their physicochemical properties were performed by several groups in order to characterize these particles. Sensitivity towards some antibiotics, factor exchangeability, and molecular weight favor the idea that mitochondrial ribosomes are of the prokaryotic type [3-51. The low RNA content and the low negative charge to mass ratio, however, oppose this assumption [6]. Elucidation of the structure of the protein moiety of the 55s ribosome should provide additional information for the classification of these particles. The method preferred for mapping 70s and 80s ribosomal proteins is two-dimensional polyacrylamide gel electrophoresis [7,8]. Ribosomal proteins from mammalian mitochondria, available in minute quantities only, were found to exhibit a lower basicity than 70s or 80s ones. With the introduction of isoelectric focusing the separation of proteins in the first dimension could be improved by replacing the molecular sieve effect and increasing the resolving power. In this paper we describe a method which combines isoelectric focusing with flat-gel disc electrophoresis in the second dimension for mapping ribosomal proteins. For mitochondrial ribosomal proteins this method yielded 60 dark plus 47 weak protein spots with isoelectric points ranging from 4.0 to 9.5. The amount of sample needed could be reduced to 50 pg of protein. 2. Experimental

Embryonic development and mitochondrial function

SummaryInhibition of mitochondrial protein synthesis in rat embryos during late organogenesis leads to impaired embryonic development.1.Thiamphenicol (TAP), similar to chloramphenicol, inhibits in vivo the synthesis of cytochrome oxidase (cytox), which is partially synthesized by the mitochondrion. Subsequently, DNA synthesis and embryonic growth are affected.2.Embryos on day 10 and 11, in contrast to embryos on day 9 of gestation, show a high sensitivity of mitochondrial protein synthesis, measured as cytox activity. From day 10 onwards, such an inhibition leads to pronounced impairment of DNA synthesis. The rat hemochorial placenta starts functioning on day 12 of gestation. Larger doses of TAP are required to inhibit cytox and DNA synthesis for treatment after placentation rather than before placentation.3.Dose-response relationships differ depending on the date and duration of treatment. Application of TAP for 1 day requires 10–30 mg/kg TAP to inhibit cytox synthesis and 60–100 mg/kg to impair embryonic growth. Prolongation of treatment to 4 days (day 10–13) lowers the dose required for inhibition of DNA synthesis to 10 mg TAP/kg/day. This is lower than the human therapeutic dose. Larger doses lead to embryolethality.4.The extent of inhibition of DNA synthesis provoked by inhibition of mitochondrial protein synthesis depends on a number of factors which include: different growth rates during organogenesis, the number of mitochondria present prior to treatment, availability of extramitochondrial ATP sources and placental permeability barrier.

Thiamphenicol during the first trimester of human pregnancy: Placental transfer in vivo, placental uptake in vitro, and inhibition of mitochondrial function☆

Thiamphenicol (TAP) was administered as a single oral dose of 500 mg (8–9 mg/kg) to pregnant women about to undergo therapeutic abortions. The length of gestations varied from 7 to 12 weeks. The concentrations of TAP were determined by electron capture gas chromatography in maternal serum, placenta, amniotic fluid, and various embryonal/fetal tissues when abortion occurred 1.5 to 20 hr after drug administration. TAP was rapidly transferred across the placental membranes and was fairly evenly distributed in the tissues of the embryonal/fetal compartment. The placental concentrations of TAP were usually higher than the corresponding maternal serum levels (1.1 to 7-fold), suggesting some active role of the placenta in TAP uptake. This was confirmed by in vitro studies with immature and term placenta tissue. From a bathing medium containing 10 μg/ml (comparable to therapeutic blood levels) TAP was accumulated in the intracellular compartment to concentrations about 2.6 times those in the medium. This process was time and temperature dependent. Inspite of the low content of mitochondrial particles in human fetal liver the relatively few mitochondria present exhibited high protein synthesis in vitro which decreased with increasing gestational age. Mitochondrial protein synthesis was inhibited by TAP, and a 50% reduction of amino acid incorporation was observed at 10–15 μg TAP/ml. Such levels of the drug are required for successful chemotherapy. To ascertain such therapeutic drug levels, TAP is usually given at 3 g daily (divided, e.g., into three doses 1 g each). The data obtained for a single application of 0.5 g TAP suggest inhibition of mitochondrial protein synthesis during therapy. While the pharmacokinetic characteristics of TAP suggested that this compound might be well suited to treat intrauterine bacterial infections sensitive to TAP, the drug concentrations reached in human embryonal tissues in vivo are known to produce embryolethality in pregnant rats at comparable developmental stages. The findings reported here raise serious concerns about the suitability of TAP for repeated administration to pregnant women during the first trimester of gestation.

Chloramphenicol/thiamphenicol and cycloheximide as tools for the measurement of mitochondrial protein synthesis in vitro during organogenesis of rat embryos

Summary1.A test system was developed to allow the measurement of protein synthesis in vitro in mitochondria from tissues which were accessible only in small quantities. The subcellular fractions which could be isolated are not purely mitochondrial but contain other particles as well, mainly microsomal, which are also active in protein synthesis. The following differences between mitochondrial and microsomal protein synthesis in vitro were used to measure selectively the mitochondrial portion in cell fractions sedimenting between 600 and 10000×g:- selective inhibition of mitochondrial protein synthesis by chloramphenicol/thiamphenicol- selective inhibition of microsomal protein synthesis by cycloheximide- kinetics of amino acid incorporation- a medium favoring mitochondrial protein synthesisActivity of mitochondrial protein synthesis was based on measurements of cytochrome oxidase, a mitochondrial marker enzyme.2.The technique developed was used for the evaluation of mitochondrial protein synthesis in mammalian embryonic tissues. It may equally well be applied to other tissues available in small amounts and in cases where the isolation of highly purified mitochondrial fractions is met with difficulty.3.Comparing the rate of 14C-phenylalamine incorporation into mitochondrial protein from rat embryos at different stages of gestation, it was found that mitochondria from 11-day-old rat embryos exhibit an approximately 30-fold higher capacity for protein synthesis than those of day 13–16. On day 12 the capacity is 6 times higher than on the following days. The results presented supply evidence for the special role of the mitochondrial function during organogenesis, especially at the time of placentation. Drugs, capable of inhibiting mitochondrial protein synthesis, are expected to have a marked effect on embryonic development.

Effect of chloramphenicol infusion on the rate of synthesis of cytochrome oxidase in mammalian embryonic tissue

Chloramphenicol (CAP) is known to interfere with protein synthesis of bacteria at the ribosomal 50-S-subunit, whereas i t does not effect prorein synthesis at the 80-S-ribosomes of mammalian species. But chloramphenieol-sensitive protein synthesis has been shown to exist in mammalian mitoehondria (Roodyn et al., 1961). A typical membrane-bound mitochondrial enzyme is cytochrome oxidase (Cytox). Since embryonic tissue at the stage of organogenesis is growing at a very fast rate, we became interested in investigating the result of an interference of CAP with the synthesis of embryonic Cytox. The correlation between the synthesis of an important mitochondrial component and growth and differentiation processes seems to be an interesting problem. Therefore, in addition to biochemical experiments we also performed teratological studies. An inhibition of the synthesis of Cytox by CAP has been shown by Firkin and Linnane (1969) and by Kroon and De Vries (1969) who used partial hepateetomy as a model. But the growth rate of regenerating fiver is several orders of magnitude smaller when compared with embryonic growth. After conventional modes of application CAP is metabolised in the liver and e]iminated at a very fast rate. Therefore, a satisfactory serum level couldbe observed only after partial hepateetomy (DcVries and Kroon, 1970). In our experiments CAP was administered by a new technique of continuous intravenous infusion without immobilisation of the animals. Results of these experimental series have been presented at two meetings (Oerter et al., 1970a; Oerter et al., 1970b).

Some aspects of teratogenesis and mutagenesis in mammalian embryos.

Mammalian embryonic tissue is an especially sensitive target for attack by a variety of drugs and environmental substances. Therefore, elucidation of the mechanism of toxic action at the molecular and cellular level is of considerable interest. Since developmental processes are complex, there is a need for simpler model systems that allow the study of toxic action at this level. The model system should be complex enough to include typical morphogenetic events. Such a system is presented in which the differentiation of limb buds of embryos (mice, rats, rabbits, etc.) from the blastema stage to recognizable cartilaginous bone anlagen representing scapula, humerus, ulna, and radius and the hand skeleton can be studied. This system is suitable for studying chondrogenesis and differentiation of cartilage at the cellular as well as the morphogenetic level, under both normal and pathological conditions. The induction of clear‐cut “malformations” is possible in vitro. Some pharmacokinetic aspects are also discuss...

Einflu von Cyclophosphamid auf die RNS-Synthese in Zellkernen und Mitochondrien

Substanz P wurde bisher nach dem Verfahren yon GADI)UM (1964) extrahiert, das wie alle frfiheren Verfahren mit saurem Kochen des Gewebes beginnt. Wir fanden die Substanz P-Aktiviti~t in der Proteolipidfraktion yon Hirngewebe bei Extraktion nach FoLcg u. LEES (195 l), wobei die aktive Fraktion durch saures Kochen vom Lipid getrennt wurde. Nach Chloroform-Methanol-Extraktion ist im Geweberiickstand keine Substanz P mehr vorhanden.

Untersuchungen über die Zellkern- und Mitochondrien-DNS nach Markierung mit H3-bzw. C14-Thymidin

Die Zunahme des Leberfettgehaltes nach einmaliger Alkoholverabreichung wird auf die vermehrte Mobilisierung yon Depoffett zuriickgefiihrt 1. Zur K1/irung der Frage, ob auch ffir die Entstehung der Fett]eber nach chronischer Alkoholverabreichung eine verst/irkte Mobilisierung yon Depotfett eine Rolle spielt, untersuchten wir an weiBen M/iusen, die 6 Monate lang anstelle yon Trinkwasser eine 15 °/oige (w/v) AlkohollSsung erhielten, den Fettgehalt der Leber sowie den Gehalt des Serums an freien (FFS) und veresteren (EFS) Fetts/~uren. ~ach 6monatiger Alkoholzufuhr waren der Leberfettgehalt und der Gehalt des Serums an FFS und EFS erhSht. Die vermehrte Angabe yon FFS aus den Depots bewirkt in der Leber eine Stimulierung der Triglyceridsynthese. Die vermehrt gebildeten Trig|yceride werden tells in der Leber gespeichert, tells an das Blur abgegeben und ffihren dort zum Anstieg der EFS. Dutch Behandlung der alkoholisierten Tiere mit dem fl-Adreno]yticure 1-(Isopropylamino)-3 (m-toloxyl)-2-propanol ( = K 5 5 9 2 ) 24Std lang vor dem T5ten kommt es zur Abnahme der erhShten Gehalte an Fett in der Leber und an EFS im Serum. Es wird geschlossen, dab auch an der Entstehung der Fettleber nach chronischer Alkoholverabreichung eine verst/~rkte Mobilisierung yon Depotfett, und zwar durch die durch Alkohol vermehrt freigesetzten Kateeholamine 2 wesentlich beteiligt sein dtirfte.

TRANSFER OF 2,3,7,8-TETRACHLORO-DIBENZO-p-DIOXIN (TCDD) TO THE MOUSE EMBRYO, FETUS AND NEONATE

ABSTRACT 1) Following administration of TCDD to pregnant mice (5, 12.5 or 25 μg/kg by oral, s.c. or i.p. route), very low TCDD levels were found in the embryo/fetus between days 11 – 18 of gestation. Prior to placentation (day 9) higher TCDD levels were found in the embryo than from day 11 on. 2) First attempts were made to correlate toxicokinetic data with embryotoxic effects (cleft palate and weight reduction). 3) TCDD is efficiently transferred via mothers milk. Neonates nursed by TCDD-treated mothers received, on a body weight basis, doses of TCDD which approached those administered to their mothers.