Bacterial mutagenicity investigation of epoxides: drugs,drug metabolites,steroids and pesticides

Although it has been observed that many epoxides are ultimate mutagens, surprisingly little is known about epoxides to which man may be extensively exposed, e.g., physiological compounds, drugs, drug metabolites and pesticides. We have now investigated 35 such and related epoxides for mutagenicity, using reversion of his^?Salmonella typhimurium TA98 and TA100 as biological end-point. None of the tested steroids (12 compounds), vitamin K epoxides (3 compounds) and pesticides (dieldrin, endrin, HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a5,6,7,8,8a-octahydro-1,4-methanonaphthalene), heptachlor epoxide) showed any mutagenic activity. Negative results were also obtained with the antibiotics oleandomycin, anti-capsin and asperlin, the cardiotonic drug resibufogenin, the widely used parasympatholytic drugs butylscopolamine and scopolamine, the sedatives valtratum, didovaltratum and acevaltratum, the tranquilizer oxanamide as well as with the drug metabolites carbamazepine 10,11-oxide and diethylstilbestrol α,β-oxide. Three barbiturate epoxides, formed by metabolism of allobarbital, alphenal and secobarbital, caused weak but reproducible mutagenic effects at high concentrations. The cytostatic agent ethoglucide was the only drug having substantial mutagenic activity. Its mutagenic potency was similar to those of the control epoxides styrene 7,8-oxide, p-bromostyrene 7,8-oxide and m-bromostyrene 7,8-oxide, but much lower than those of benzo[a]pyrene 4,5-oxide, benzo[e]pyrene 4,5-oxide and 7,12-dimethylbenz[a]-anthracene 5,6-oxide.Some epoxides were also tested in other Salmonella typhimurium strains or in the presence of rat-liver S9 mix. Positive results were only obtained with compounds that had already been detected as mutagens in the direct test with strain TA100.     

Speicherung und mobilisierung von Kohlenhydraten und Lipiden in der Blattwespe Gilpinia hercyniae

Von Larven (drei Populationen, L₃, L₄-, L₄, L₅- und alte L₅-Larven), adulten Weibchen vor (I_v) und nach (I_n) Eiablage, Kokons (K) und Exuvien (E) wurden die Konzentrationen von löslichen Kohlenhydraten, Alanin, Prolin, Glykogen und Lipiden sowie die Frisch- und Trockengewichte bestimmt. Aus den Ergebnissen werden Rückschlüsse auf den Energiestoffwechsel der Entwicklungsstadien Embryo, Larve und Imago gezogen.

---

Summary The concentrations of soluble carbohydrates (Fig. 2), alanine and proline (Fig. 3), glycogen and lipids (Fig. 4) and the weights (Fig. 1, Tab. I) of larvae (three populations, L₃, L₄-, L₄, L₅- and old L₅-instar larvae), adult females before (I_v) and after (I_n) oviposition, cocoons (K) and exuviae (E) (Tab. II) were determined. Embryonic, larval and adult stages of development have characteristic types of energy metabolism. The lipid catabolism of the embryonic stage is substituted during larval development by anabolic pathways based on resorbed sugars absorbed from spruce needles. During the first larval stages these soluble carbohydrates are directed to a glycogen pool in relatively large amounts. This pool is only slightly enlarged whereas lipids are synthesized in relatively large amounts between the 14th and 28th day after oviposition. Metamophosis is correlated with a decrease of glycogen. Lipids are used as an energy source by imagines and in eggs. Energy is distributed in the body by movement of trehalose in the larval stage and by a proline-alanine-shuttle in the adult stage.

     

Characterization of a β-glucosidase from Glycine max which hydrolyses coniferin and syringin

A β-glucosidase which rapidly hydrolyses the cinnamyl alcohol glucosides coniferin and syringin has been purified from cell cultures, hypocotyls and roots of Glycine max. Isoelectric focusing in a column separated the enzyme from several other β-glucosidases which were inactive against either substrate. Syringin and coniferin were the best substrates tested. Both exhibited identical V_max values, whereas the K_m of coniferin (0.6 mM) was twice that of syringin (0.3 mM). The widely used synthetic substrates 4-nitrophenyl-β-glucoside and 4-methyl-umbelliferyl-β-glucoside were poorly utilized. Glucono-1,5-lactone was an effective competitive inhibitor with a K_i of 0.01 mM. From the observed-substráte specificity, a role in the lignification process of higher plants may be predicted for this β-glucosidase.     

The leech photoreceptor cell: ultrastructure of clefts connecting the phaosome with extracellular space demonstrated by lanthanum deposition

Summary When the extracellular space in eyes of Hirudo medicinalis was traced by means of lanthanum deposition, clefts were found which extend into receptor cells connecting the phaosome vacuoles with the extracellular space. The membrane of the phaosome, which bears the photoreceptor microvilli, is continuous with the external membrane of the receptor cell. Thus mechanisms by which the initial events of photoreception bring about electrical events at the cell surface need not differ from those being considered for other photoreceptor cells.Intracleft structures were revealed in negative constrast by the lanthanum deposits. Bridges join the opposed membranes on either side of a cleft. Isolated isodiametric profiles (120 A in diameter), and zig-zag linear structures, that are perhaps linear arrays of the isodiametric structures, were revealed in tangential sections of lanthanum filled clefts.Supported by the National Science Foundation, Grant GB-4822, and by the Deutsche Forschungsgemeinschaft.We thank Mrs. Carol Deuel Sundeen for technical assistance.     

Synthese von Speicherstoffen aus Pyruvat durch Rhodospirillum rubrum

Zusammenfassung Pyruvat kann durch belichtete Zellen von R. rubrum assimiliert und zur Synthese verschiedener Zellbestandteile verwendet werden. Ist das Angebot an Substrat und Lichtenergie groß genug, so wird ein Teil der Brenztraubensäure in Speicherstoffe eingebaut. Suspension der Zellen in Phosphat-puffer und Inkubation unter Wasserstoff stimuliert die Bildung des Speicherstoffes Poly--Hydroxybuttersäure. Die Synthese eines ebenfalls als Speicherstoff in R. rubrum bekannten Polysaccharids konnte vor allem bei großem Substratüberfluß und Inkubation unter Stickstoff beobachtet werden; es wurde jedoch auch unter optimalen Bedingungen nur ein geringer Anteil der total aufgenommenen Brenztraubensäure zur Synthese dieses Speicherpolysaccharids verwendet.

---

Synthesis of storage material from pyruvate by Rhodospirillum rubrum

Summary Illuminated cells of R. rubrum assimilate pyruvate and use it for the ynthesis of different cell components. A sufficient supply of substrate and light energy provided, part of the pyruvate is built into storage products. If the cells are suspended in phosphate buffer and kept under an atmosphere of molecular hydrogen the formation of the -hydroxybutyrate polymer is stimulated. An excess of substrate and incubation under molecular nitrogen leads to the synthesis ofa polysaccharide, also known as storage product in R. rubrum; but even under optimal conditions only a small part of the pyruvate is used for the synthesis of this storage polysaccharide.

     

Untersuchungen zur lichtinduzierten ATP-Pa-Austauschreaktion in Spinatchloroplasten

Zusammenfassung Bei Belichtung bildet sich in Chloroplasten ein Energie-pool, der im Dunkeln innerhalb von 20–30 min wieder geleert wird. Dabei kann ein Austausch zwischen ATP und anorganischem Phosphat stattfinden. Die Austauschreaktion ist abhängig von Mg²⁺ und durch ADP nicht hemmbar; dagegen blockieren NH ₄ ⁺ -Ionen. Optimaler pH-Wert in Licht- und Dunkelphase ist pH 7,5–8.Der energiereiche Zwischenzustand bildet sich nur bei Anwesenheit von DTT im Licht; Zugabe von Phosphat im Licht hat keinen Einfluß auf seine Entstehung, erhöht jedoch seine Stabilität im Dunkln. Es wird diskutiert, wie sich die energiereichen Zwischenzustände bei der lichtinduzierten ATP-P_a-Austauschreaktion und bei der lichtinduzierten ATP-Synthese zueinander verhalten im Hinblick auf deren Bedeutung für die chemiosmotische Phosphorylierungshypothese.

---

Studies on the light-induced ATP-P_i exchange reaction with spinach chloroplasts

Summary Light induced the formation of an energy pool in chloroplast fragments which was emptied within 20–30 min in the dark, while an ATP-P_i exchange was going on.The exchange reaction was dependent on Mg²⁺ and was inhibited by NH ₄ ⁺ but not by ADP. The optimum pH in the light and in the dark stage of the reaction lay between pH 7.5 and 8.The high energy intermediate was formed only in the presence of DTT in the light stage; phosphate had no influence on the formation of the intermediate in that period, but increased its stability in the dark.The relation between both high energy intermediates, that of the light-induced ATP-P_i exchange reaction and that of the light-induced ATP synthesis, is discussed in its meaning for the hypothesis of chemiosmotic phosphorylation.

     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

Blood serum proteins and the mineralization of bone ground substance

Summary Direct tracing experiments with fluorochrome-labeled homologous blood serum show that certain serum components are taken up by the bone substance of young and adult rats. These proteins are concentrated and incorporated into the organic matrix while it is being formed. They retain their fluorescent label for long periods of time and withstand histological fixation and decalcification. In experimental rickets, no labeled serum protein is seen to be incorporated into the uncalcified rachitic osteoid. Its uptake begins, however, concomitantly with the onset of mineralization during the healing period. The results of experiments are interpreted in terms of a calcium-carrying serum protein being complexed and precipitated by osteoblast products to form an essential component of apposition zones which, possibly, initiates nucleation and the subsequent steps of calcification. Fractionation experiments to define the serum component(s) involved in this process, will be continued. So far, they have resulted in a fraction containing albumin, ₁-macroglobulin, transferrin, and haptoglobin.

---

Zusammenfassung Gewisse Anteile von Fluorochrom-markiertem homologem Blutserum lassen sich nach parenteraler Verabreichung in der Knochensubstanz junger und erwachsener Ratten direkt nachweisen. Diese Serumproteine werden in hoher Konzentration während der Knochenbildung in die organische Matrix eingebaut. Sie behalten ihre Markierungsfluoreszenz für lange Zeit und widerstehen der Fixierung und der Entkalkung. Während experimenteller Rachitis ist eine Aufnahme dieser markierten Serumproteine in das unverkalkte rachitische Osteoid nicht festzustellen. Ihre Inkorporation beginnt jedoch wieder gleichzeitig mit dem Einsetzen der Verkalkung, wenn die Rachitis zur Ausheilung gebracht wird. Die experimentellen Resultate deuten darauf hin, daß ein calciumtragendes Serumprotein mit gewissen Produkten der Osteoblasten Komplexe bildet, dabei präzipitiert und zu einem Bestandteil der Appositionssäume wird, der möglicher Weise die Verkalkung der organischen Knochengrundsubstanz in Gang setzt. Fraktionierungsexperimente zur Bestimmung der an diesem Vorgang beteiligten Serumkomponente(n) sind noch nicht abgeschlossen. Sie halten derzeit bei einer Fraktion, die Albumin, -1-Makroglobuline, Transferrin und Haptoglobin enthält, und werden weiter fortgesetzt.

---

---

Supported by United States Public Health Service Grant No. AM06705.