Gert L. Laqueur

Transplacental Chemical Induction of Microencephaly in Two Strains of Rats. I

Summary An experimental model for successful chemical induction of microencephaly is described. Methylazoxymethanol, the aglycone of cycasin, when administered to rats at a dose of 20 mg/kg of body weight on day 14 or 15 of pregnancy, uniformly produces microencephaly in all litter mates. This effect occurs independent of the strain of rats used and is highly reproducible. The condition is not associated with microsplanchnia and appears to be limited to the cortical hemispheres of the brain. Preliminary pathologic studies indicate a marked cytotoxic effect of the chemical on the neuroblast. Gliomas have occurred in about 10% of microencephalic rats permitted to live beyond 1 year.

The induction of intestinal neoplasms in rats with the glycoside cycasin and its aglycone

Experiments are described dealing with the carcinogenic effects of (1) the crude cycad seed material, (2) the glycoside, cycasin, aβ-D-glucosyloxyazoxymethane, isolated from the crude material, and (3) the first metabolic breakdown product of cycasin, the aglycone of cycasin or methylazoxymethanol, on the intestinal tract of rats. While the crude material and cycasin produced tumors exclusively located in the large intestine, the aglycone when injected intraperitoneally, resulted in neoplasms of the small intestine as well. Supportive experiments in germfree animals are cited which indicate that cycasinper se, whether present in the intestinal tract or absorbed and excreted, is innocuous. Apparently, the glycoside is converted in conventional rats into the highly toxic aglycone through aβ-glucosidase of bacterial origin. Hence, it would seem likely that the carcinogenicity of the crude cycad material and of cycasin depends on a bacterial flora capable of providing the enzyme necessary for the liberation of the aglycone. Während Cycasin sowie seine Muttersubstanz, das Endosperm derCycas circinalis, ausnahmslos Tumoren im Dickdarm hervorriefen, gelang es mit dem Aglykon nach intraperitonealer Injektion auch Tumoren im Duodenum zu erzeugen. In keimfreien Tieren ist das Cycasin auch in sehr großen Dosen wirkungslos, wird aber von der Darmwand absorbiert und von den Tieren zum größeren Teil ausgeschieden. Dem keimfreien Tier fehlt die Glucosidase, die normalerweise das Cycasin in sein sehr toxisches Aglykon und Zucker spaltet. Die Möglichkeit besteht deshalb, daß eine erfolgreiche Krebserzeugung mit Cycasin an eine Darmflora gebunden ist, die das notwendige Enzym für die Spaltung liefern kann.

Intellectual Deficit Associated with Transplacentally Induced Microcephaly in the Rat

Fischer rats injected with methylazoxymethanol late in pregnancy produce young with considerably reduced cerebral hemispheres. They appear normal otherwise. As adults these animals make many more errors in the Hebb-Williams maze than do control animals.

QUANTITATIVE CHANGES WITH AGE IN THE DNA CONTENT OF METHYLAZOXYMETHANOL-INDUCED MICROENCEPHALIC RAT BRAIN

Abstract— DNA synthesis in methylazoxymethanol (MAM)‐treated foetal brain was reduced during the first 3 days after the injection of the compound into the mother rat. The MAM‐treated brain grew at almost the normal rate after this period, but the reduction in DNA persisted through maturity of the animal. This difference in DNA content between normal and microencephalic brain was restricted to the cerebral hemispheres. The major increase in DNA content of prenatal brain occurred in the cerebrum, whereas the postnatal increase took place in the cerebellum. jH‐Labelled MAM was incorporated more extensively into foetal brain DNA than into RNA. The half‐life of the MAM‐modified nucleic acids was 4–5 days. We suggest that removal of necrotic cells from the brain may account for the rapid loss of label from nucleic acids.

Role of intestinal microorganisms in determining cycasin toxicity.

Summary The role of intestinal bacteria in converting the naturally occurring glucoside cycasin to its hepatotoxic and carcinogenic aglycone (methylazoxymethanol) has been studied. Germfree rats, in which cycasin is nontoxic, were monocontaminated with several strains of bacteria prior to being given cycasin. Levels of glucosidase activity in the bacteria were determined by the assay of cell free extracts, using cycasin as a substrate. The toxicity of cycasin in rats given the various bacteria and the amounts of unchanged cycasin excreted were consistent with the glucosidase assays. Intestinal microorganisms therefore convert cycasin to the toxic aglycone, and variations in the intestinal flora probably have a role in determining the toxicity of ingested cycasin.

Cycasin Excretion in Conventional and Germfree Rats

Summary Urinary and fecal excretion of the naturally occurring toxic and carcinogenic glycoside, cycasin, β-D-glycosyloxyazoxymethane, was determined in germfree and conventional Sprague-Dawley male rats. Excretion in germfree rats was nearly quantitative while only 18 to 35% was recovered in conventional rats. Since cycasin is inert until acted upon by a β-glucosidase, the lack of enzymatic cleavage in germfree rats suggests the bacterial flora as the most likely source for the enzyme in conventional rats. Considerable variation was encountered in the amount of cycasin which was not recovered and was presumably metabolized in conventional rats. The possible significance of this variability is discussed in relation to toxicity and carcinogenicity.

Evidence for Transplacental Passage of the Natural Carcinogen Cycasin and Its Aglycone

Summary The passage of cycasin and MAM through the placenta and mammary gland has been investigated in rats during pregnancy and lactation, respectively. Both cycasin and MAM cross the placenta when cycasin is given orally to pregnant rats. Both compounds can also pass the mammary gland and are excreted with the milk during lactation provided that they are administered within 1 day before littering or during lactation. MAM has also been found in the fetuses after its intravenous injection into the pregnant golden hamster. The recovery of MAM from the fetuses of rats and hamsters supports a direct action of the carcinogen and teratogen on fetal tissues.

Effect of Hypophysectomy on Electrolyte Excretion in Dogs with Ascites Produced by Thoracic Inferior Vena Cava Constriction

Following complete or nearly complete hypophysectomy in dogs with thoracic inferior vena cava constriction, a striking increase in sodium (Na) excretion occurred concomitant with a fall in femoral venous pressure. The previously high venous pressure and renal Na retention were restored by administration of thyrotropin (TSH), growth hormone injection or further constriction of the thoracic inferior vena cava, but adrenocorticotropic hormone (ACTH) was without effect on these functions. The data indicate that pituitary hormones influence the accumulation of ascitic fluid by contributing to the maintenance of an adequate level of venous hypertension.

The influence of the central nervous system on metabolism and endocrine activity as based on transection of the brain stem in dogs

This report deals with the effect of midbrain transection on the metabolic balance of nitrogen, sodium and potassium and the urinary excretion of creatine, creatinine and formaldehydogens in 10 female dogs. Similar studies have been carried out on 3 control dogs subjected to hemispherectomy, hemidecortication and unilateral occipital lobectomy respectively. The dogs with transections of the brain stem showed a trend toward a negative nitrogen balance. This was especially marked in 4 of the animals; in 2 other animals the balance shifted from a distinctly positive to a weakly negative balance. The animals which survived more than one month postoperatively showed a return to a positive nitrogen balance by the third postoperative week. Two of the animals manifested no change in nitrogen balance. No correlation could be found in the differences in nitrogen balance and the site and degree of brain stem transection. In the control animal with a hemispherectomy and in which there were no complications from infection, no change in nitrogen balance occurred postoperatively. The potassium balance resembled the nitrogen balance in almost every instance. The sodium balance became positive postoperatively in both the experimental and control dogs. This retention of sodium persisted 10 to 14 days. There was no increased urinary excretion of adrenal steroids as measured by formaldehydogens after either transection, hemispherectomy or hemidecortication. ACTH and cortisone produced an increase in urinary formaldehydogens when given in high doses. Creatinuria occurred in all animals with transection of the brain stem and lasted 10 to 25 days. The control dog with a hemispherectomy showed no creatinuria; the control dog with an infection after hemidecortication had creatinuria for 3 days during the height of stet OK infection. In the animals with brain stem transection there was an abrupt decrease in the urinary creatinine on the day of operation. This persisted as long as the animal lived. The decrease did not occour following hemidecortication or hemispherectomy. There was an impaired glucose tolerance following brain stem transection, but no change in insulin sensitivity. No changes were found in the controls. There are few clues as to the nature of these disturbances. A speculative interpretation is presented. Dieser Bericht handelt von der Wirkung der Mittelhirndurchtrennung auf den metabolischen Ausgleich von Stickstoff, Natrium und Kalium und auf die Ausscheidung von Kreatin, Kreatinin und Formaldehydogenen auf dem Harnweg bei 10 weiblichen Hunden. Gleiche Studien wurden an 3 Kontrollhunden durchgeführt, die einer Hemisphärektomie, einer Hemidekortikation bzw. einer einseitigen okzipitalen Lobektomie unterzogen worden waren. Die Hunde mit Gehirnstammdurchtrennung zeigten eine Neigung zu einem negativen Stickstoffausgleich. Dies war bei 4 Tieren besonders ausgeprägt; bei zwei anderen Tieren verschob sich der Ausgleich von einem deutlich positiven zu einem schwach negativen. Die Tiere, die die Operation um mehr als einen Monat überlebten, zeigten eine Rückkehr zu einem positiven Stickstoffausgleich um die 3. Woche nach der Operation. Zwei Tiere zeigten keine Änderung im Stickstoffausgleich. Es konnten keine Beziehungen zwischen den Unterschieden im Stickstoffausgleich und dem Ort und Grad der Gehirnstammdurchtrennung gefunden werden. Bei dem Kontrolltier mit Hemisphärektomie, bei dem keine Komplikationen durch Infektion auftraten, erfolgte nach der Operation keine Änderung im Stickstoffausgleich. Der Natriumausgleich glich dem Stickstoffausgleich in beinahe allen Fällen. Der Natriumausgleich wurde nach der Operation sowohl bei den dem Versuch unterzogenen Hunden als auch bei den Kontrollhunden positiv. Diese Natriumretention dauerte 10 bis 14 Tagen an. Es trat keine vermehrte Ausscheidung adrenaler Steroide auf dem Harnweg ein, gemessen an den Formaldehydogenen entweder nach der Durchtrennung oder nach der Hemisphärektomie oder der Hemidekortikation. ACTH und Cortison bewirkten eine Vermehrung der Harn-Formaldehydogene, wenn sie in starken Dosen gegeben wurden. Kreatinurie trat bei allen Tieren mit durchtrenntem Gehirnstamm ein und dauert 10 bis 25 Tage an. Der Kontrollhund mit Hemisphärektomie zeigte keine Kreatinurie; der Kontrollhund mit einer Infektion nach Hemidekortikation hatte eine Kreatinurie durch 3 Tage während des Höhepunktes der Infektion. Bei den Tieren mit Gehirnstammdurchtrennung trat am Tag der Operation eine plötzliche Abnahme des Harn-Kreatinins ein. Diese dauerte an, so lange das Tier lebte. Die Abnahme trat nach Hemidekortikation oder Hemisphärektomie nicht ein.

Gnotobiotes as Useful Tools in Studies in Carcinogenesis with Cycasin, a Natural Glucoside

Experimental studies in our laboratory have established marked oncogenic properties of the glucoside cycasin, β-D-glucosyl-oxyazoxymethane. The glucoside is extractable from seeds or tubers of ancient gymnosperms belonging to the family of Cycadacea. This plant was widely distributed during the mesozoic period, but todays members are found only in tropical and subtropical parts of the world. Starch prepared from seeds and tubers are used by many people as a source of food. It was apparently known to these populations for an unknown period of time that the crude starch contained a poison, and similar methods for removal of the poison have been practiced by them. The interested reader is referred to an excellent review of the toxicity of cycads by Marjorie Grant Whiting covering what was known about cycad toxicity up to 1963 (1).