No abstract

To determine whether the release of acrolein from oxazaphosphorinane-cytostatics contributes to their cytotoxic action, the effect of 4-hydroperoxycyclophosphamide, 4-hydroperoxy-semi-cyclophosphamide, 4-hydroperoxy-dechloro-cyclophosphamide, and acrolein on murine L 1210 leukemia cells in vitro was compared by measuring the median survival time (MST) after transplantation of the tumor cells in DBA2/Han mice. We found that only 4-hydroperoxycyclophosphamide, which is able to release both acrolein and the alkylating metabolite phosphoramide-mustard, decreased the transplantability of L 1210 cells, while the structurally analogous 4-hydroperoxy-dechloro-cyclophosphamide and 4-hydroperoxy-semi-cyclophosphamide, which under physiological conditions only release acrolein but no alkylating split products showed no cytotoxicity. Acrolein itself showed only a marginal effect, when administered in concentrations equivalent to the release of acrolein from the oxazaphosphorinane-derivatives in test. In this case, however, significant lysis of the L 1210 cells was observed by estimating dye exclusion, while acrolein released intracellularly from 4-hydroperoxy-oxazaphosphorinane-compounds did not. This points to a different mechanism of the cytotoxic action of extracellular acrolein and acrolein released intracellularly from activated oxazaphosphorinane-compounds. The results suggest that the cytotoxic effect of activated cyclophosphamide is based on the alkylating moiety of the molecule. Neither the 4-hydroperoxy-group nor the activated oxazaphosphorinane-ring itself, nor acrolein released intracellularly during toxification of activated cyclophosphamide exert a direct cytotoxic effect. Thus, the release of acrolein from activated CP apparently does not contribute to the cytotoxicity of CP in vivo.

Previous studies suggested that increased blood levels of, or increased tissue sensitivity to, glucocorticoids may contribute to catabolism in acute uremia. To examine this possibility we determined urea nitrogen (urea-N) appearance, plasma levels of N^l-methylhistidine and the activity of the alkaline myofibrillar proteinase in acutely uremic rats with and without treatment with RU 38486, a selective antiglucocorticoid. Forty-eight hours after bilateral nephrectomy, the rats had markedly elevated serum levels of urea-N, creatinine, potassium and phosphorus. In uremic rats receiving RU 38486, comparable levels of serum creatinine were found, but the serum levels of urea-N (221 ± 4 vs. 259 ± 5 mg/dl) and phosphorus (6.5 ± 0.3 vs. 8.5 ± 0.4 mmol/l) were significantly decreased as compared to uremic animals without RU 38486. In comparison to sham-operated rats, urea-N appearance (net urea production) was increased by 56% 48 h after bilateral nephrectomy. This increment was almost completely reversed in uremic animals receiving the antiglucocorticoid. In untreated uremic rats, plasma levels of N_tethylhistidine were 10.3 ± 0.9 μg/dl, whereas the administration of RU 38486 caused a significant decline in the levels of this amino acid (7.6 ± 0.5 μg/dl). This reduction in N’-methylhistidine was associated with a concomitant decrease of myofibrillar proteinase activity in muscle tissue homogenates. Compared to sham-operated animals, this proteinase activity was increased by 30% in uremic rats, but was normal in those given RU 38486. Taken together, these data support the view that in acute uremia accelerated ureagenesis occurs, while enhanced muscle protein breakdown, owing to an increment in myofibrillar proteinase activity, provides the necessary amino acid precursors. Furthermore, the activity of this proteinase, and therefore the rate of muscle protein breakdown, strongly depend on the presence or absence of glucocorticoids in uremia.

The effect of adrenalectomy on the enhanced protein degradation in acute uremia was investigated. Therefore, serum urea nitrogen, urea N appearance and N^t-methylhistidine were followed in bilaterally nephrectomized rats. At 48 h after induction of uremia the animals displayed serum urea nitrogen levels of 223 ± 9.5 mg/dl as compared to 26.0 ± 1.0 mg/dl in sham-treated rats. This increment was significantly attenuated in acutely uremic, adrenalectomized animals (176 ± 6.0 mg/dl). When these rats were substituted with corticosterone (5 mg/kg body weight), serum urea nitrogen readily increased to levels of acutely uremic animals with intact adrenal glands (225 ± 6.0 mg/dl). The net generation of urea, as determined by the urea N appearance, was significantly increased during acute uremia (370 ± 26 mg/48 h) as compared to SHAM animals (220 ± 15 mg/48 h). This increment of urea formation could almost be completely reversed by simultaneous adrenalectomy (238 ± 20 mg/48 h). When these rats were substituted with corticosterone, the urea N appearance rebounded to values quite comparable to acutely uremic rats with intact adrenal glands (363 ± 30 mg/48 h). To determine whether skeletal muscle proteins might serve as a source for the enhanced protein degradation in acute uremia, plasma levels of N’-methylhistidine were measured. Bilaterally nephrectomized rats had N^t-methylhistidine values of 9.6 ± 1.0 μg/ml. In acutely uremic rats without adrenal glands, N^t-methylhistidine levels were found to be significantly decreased (6.0 ± 0.4 μg/ml). Substitution of corticosterone in these animals caused a remarkable rebound of N^t-methylhistidine values (9.2 ± 0.9 μg/ml). These results establish the capability of glucocorticoids to enhance protein degradation in acutely uremic rats. Moreover, this seems to be primarily brought about by an increment in myofibrillar protein breakdown.

The hydroperoxy and several alkylthio derivatives of the antitumor agents cyclophosphamide (2-bis(2-chloroethyl)amino tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide), ifosfamide (3-(2-chloroethyl)-2-(2-chloroethylamino)tetrahydro-2H-1,3,1-oxazaphosphorine 2-oxide) and trofosfamide (3-(2-chloroethyl)-2-(bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) were characterized by electron impact and field desorption mass spectrometry. The compounds, which are stabilized derivatives of the activated hydroxylated intermediates of cyclophosphamide (ifosfamide, trofosfamide), could be identified as 4-hydroperoxy and 4-alkylthio oxazaphosphorines. The existence of diastereomers of these products was demonstrated by thin-layer chromatography and f.d. mass spectra. Derivatization with benzylmercaptan was found to be an appropriate method for the quantitative isolation and mass spectral identification of the activated metabolic intermediates of cyclophosphamide from biological material. Using this reaction, 4-hydroxycyclophosphamide and its acyclic tautomer, aldophosphamide, which are too unstable for direct identification, were detected in urine and serum of patients treated with 3H-cyclophosphamide.