Development of a 32P-postlabelling method for the detection of 1,N 2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.     

A phase I clinical and pharmacokinetic study of the dolastatin analogue cemadotin administered as a 5-day continuous intravenous infusion

Purpose: The dolastatins are a class of naturally occurring cytotoxic peptides which function by inhibiting microtubule assembly and tubulin polymerization. Cemadotin is a synthetic analogue of dolastatin 15 with potent antiproliferative and preclinical antitumor activity. This report describes a phase I study to evaluate the administration of cemadotin to adult cancer patients by a 5-day continuous intravenous (CIV) infusion. Methods: All patients had histologically confirmed refractory solid tumors. The dose was escalated from an initial level of 2.5 mg/m² (0.5 mg/m² daily) according to a modified Fibonacci algorithm. A minimum of three patients was evaluated at each dose level until the maximum tolerated dose (MTD) was established. Treatment was repeated every 21 days until patients were removed from the study due to toxicity or disease progression. Drug-related toxicities were evaluated and graded by the U.S. National Cancer Institute's Common Toxicity Criteria. A radioimmunoassay (RIA) that detected both the parent drug and its metabolites with an intact N-terminal region of the molecule was used for pharmacokinetic studies. Results: Twenty heavily pretreated patients received a total of 40 courses of cemadotin over five dose levels ranging from 2.5 to 17.5 mg/m². Reversible dose-related neutropenia was the principal dose-limiting toxicity and 12.5 mg/m² was established as the MTD. Nonhematologic toxicities attributed to the drug were moderate, and there was no evidence of the cardiovascular toxicity noted in the prior phase I studies of cemadotin given IV as a 5-min injection or 24-h infusion. There were no objective antitumor responses. Time courses of the cemadotin RIA equivalent concentration in whole blood were defined in 14 patients during the first cycle of therapy. The RIA-detectable species exhibited apparent first-order pharmacokinetics across the entire range of doses. The mean ± SD of the observed steady-state blood concentration at the 12.5 mg/m² MTD was 282 ± 7 nM (n=3). Blood levels decayed monoexponentially following the end of the infusion, with a mean half-life of 13.2 ± 4.3 h (n=14) in all patients. Mean values (n=14) of the total blood clearance and apparent volume of distribution at steady state were 0.52 ± 0.09 l/h/m² and 9.9 ± 3.3 l/m², respectively. Conclusions: The cardiotoxic effects of cemadotin were completely avoided by administering it as a 120-h CIV infusion. Thus, cardiovascular toxicity appears to be associated with the magnitude of the peak blood levels of the parent drug or its metabolites, whereas myelotoxicity is related to the duration of time that blood levels exceed a threshold concentration. Nevertheless, the data acquired during the extensive clinical experience with cemadotin requires careful examination to assess whether advancing this compound into disease-oriented efficacy studies is merited. Received: 8 November 1999 / Accepted: 28 April 2000     

Specificities in human cell-mediated cytotoxicity.

Specificity of natural cell-mediated cytotoxicity was investigated through selective reactions detected by direct cell-mediated cytotoxicity and by inhibition of cytotoxicity through competition. Assuming that target cells reacting alike in direct cytotoxicity shared common antigens, we classified 10 target cells into three groups by target antigens: TA (target antigen) 1, 2, and 3. Partial confirmation of the three groups was achieved in the cross-competition assay. The distinction of TA 1 as a group was clear but some cross-reactivity existed between TA 2 and TA 3 cells.     

Die Wirkung von Fols?ureantagonisten (Methotrexat) auf die Regeneration der Darmschleimhaut

Zusammenfassung In vergleichenden Untersuchungen wird die Wirkung der folsÄureantagonistisch wirksamen Substanz Methotrexat auf die Regeneration der Schleimhaut von Duodenum, Jejunum, Ileum, Colon ascendens und Colon descendens von MÄusen geprüft.Im Langzeitversuch findet sich eine bis zu 3 Wochen fortschreitende langsame Zottenatrophie, vor allem am Duodenum, die auf einen Zellverlust in der Population der Regenerationszone zurückzuführen ist.Bei einmaliger hochdosierter Zufuhr von Methotrexat (Stoversuch) wird die Regenerationszone geschÄdigt, wobei anfangs der Verlust regenerationsfÄhiger Zellen mit einer gesteigerten DNS-Synthese der Restpopulation beantwortet wird; über eine gesteigerte Zellneubildung ist nach 4 Tagen die Regenerationszone wieder weitgehend hergestellt. WÄhrend der Blockade der Regenerationszone ist die Auswanderung differenzierter Zellen auf die Zotten unterbrochen bzw. verzögert; nach Wiederherstellung findet sich ein beschleunigter Zellersatz. Entsprechend dem langsameren physiologischen Zellersatz am Colon ascendens und Colon descendens ist hier eine Methotrexatwirkung in Form einer vorüber gehenden Blockade nur in abgeschwÄchter Form feststellbar.

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The action of folic acid antagonists (methotrexate) on the regeneration of the intestinal mucosa

Summary Comparative studies were made of the action of Methotrexate, the folic acid antagonist, on the regeneration of the mucosa of the duodenum, jejunum, ileum, ascending and descending colon of the mouse. In chronic experiments an atrophy of villi slowly developed up to three weeks, especially in the duodenum. The atrophy was related to a loss of some of the cells of the regeneration zone. On administering a. single large dose of Methotrexate (push-experiment) the regeneration zone was damaged. Consequently, at the onset the loss of the cells capable of regenerating provoked an increased DNA synthesis of the remaining cells. After four days the regenerative zone was nearly restored by an increased renewal of cells. During the block of the regeneration zone, the migration of differentiated cells to the villi was interrupted or delayed. After restoration there was an accelerated renewal of cells. In the ascending and descending colon, where the physiological renewal of cells is slower than in other parts of the intestine, the effect of Methotrexate became apparent in a diminished form as a temporary block.

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Herrn Professor Dr. W.Büngeler zum 65. Geburtstag.

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Inaug.-Diss. München. Die Untersuchungen wurden mit Unterstützung der Deutschen Forschungsgemeinschaft durchgeführt.