The role of biotransformation in anthracycline-induced cardiotoxicity in mice.

Anthracyclines are highly efficacious antineoplastic agents but they become cardiotoxic after repeated dosing. For the major anthracycline, doxorubicin (Dox), this toxicity is thought to be associated with the formation of the 13-dihydro metabolite. Paced mouse left atria were used to assess the cardiotoxicity of Dox, 4'-epidoxorubicin (Epi), daunorubicin (Dauno) and their major metabolites. Apart from the aglycons, all compounds (1-500 microM) reduced the contractile force. To correct for differences in cellular uptake, anthracycline concentrations were determined in the atria after 1 h of incubation. IC50 values ranged from 0.33 mumol/g for 13-dihydro-Dox to 3.5 mumol/g for Dauno. The toxicities relative to Dox, i.e., the ratio of IC50,Dox/IC50,anthracycline, ranged from 0.19 for Dauno to 2.1 for 13-dihydro-Dox (the most toxic). For Dox, Epi and Dauno, the 13-dihydro metabolite had greater toxicity than the corresponding parent compound. The pharmacokinetics of Dox and Epi in the murine heart are comparable and, thus, cannot explain the reduced cardiotoxicity of Epi. However, when pharmacokinetic data of Dox and Epi in murine heart tissue were interpreted using the relative toxicity factors, Epi would be expected to be threefold less cardiotoxic than Dox, thus providing a better correlation with in vivo data. This simple pharmacological model in combination with preclinical pharmacokinetics may contribute to the prediction of the cardiotoxic potency of new anthracyclines relative to Dox.

Simple and sensitive quantification of anthracyclines in mouse atrial tissue using high-performance liquid chromatography and fluorescence detection.

Anthracyclines are very effective against soft tissue sarcomas, with cardiotoxicity being an important side effect after repeated administration. To estimate the relative cardiotoxicity of various anthracyclines and their metabolites, we developed an isolated mouse left atrium model. To relate an effect of doxorubicin, 4'-epidoxorubicin and their four main metabolites (doxorubicinol, epidoxorubicinol and the aglycons 7-deoxydoxorubicinon and 7-deoxydoxorubicinolon) to concentrations in the tissue instead of the incubation bath, a method of quantifying the anthracyclines in small tissue samples was developed. Atria were homogenized by sonication followed by extraction of the anthracyclines with methanol. The extract was directly analyzed by high-performance liquid chromatography with fluorescence detection. Recoveries for the six compounds tested ranged from 67.5% for 4'-epidoxorubicin to 100.6% for 7-deoxydoxorubinol aglycon with coefficients of variation of 2-3% at two spiked concentrations (0.1 and 1 nmol/mg of tissue). The calibration plots were linear (r2 greater than 0.996) over the concentration range tested (0.05-1 nmol/mg wet weight). The limits of detection (4-10 pmol/mg of tissue) were low enough to allow the determination of the anthracyclines at all relevant tissue concentrations.

Isolated mouse atrium as a model to study anthracycline cardiotoxicity: the role of the beta-adrenoceptor system and reactive oxygen species.

Cancer chemotherapy with anthracyclines, of which doxorubicin (DX2) is the main representative, is limited by cardiomyopathy developing in animals and patients after cumulative dosing. The toxicity is probably related to free radical formation by the anthracycline as well as its metabolites with concomitant O2.- and .OH generation resulting in lipid peroxidation and subsequent membrane damage. An in vitro model is required to investigate the individual contribution of each metabolite to cardiotoxicity. For in vivo studies, the species of choice is the mouse because it lacks the DX-induced nephrotic syndrome seen for instance in rats and rabbits. Thus, isolated mouse heart muscle was chosen as an in vitro model. To characterize the model, we used l-isoprenaline/dl-propranolol and metacholine/atropine to measure the beta-adrenergic and the muscarinic responses of (spontaneously beating) right and (paced) left atrium. Dose response curves (n greater than or equal to 4) were highly reproducible: pD2,iso = 8.0 +/- 0.3 (left) and 8.5 +/- 0.4 (right); pD2,met = 6.7 +/- 0.1 (left) and 6.2 +/- 0.3 (right). Propranolol as well as atropine behaved as competitive antagonists, with pA2-values of 8.4 +/- 0.2/8.5 +/- 0.2 (l/r) and 9.1 +/- 0.1/9.1 +/- 0.2 (l/r), respectively. These values corresponded to those obtained with other organ preparations. We tested the effect of DX in two ways: a) by measuring the direct inotropic and chronotropic effect during 60 minutes of incubation with 10-100 microM DX in the organ bath, and b) by determining the remaining beta-adrenergic response to l-isoprenaline after the incubation period. Both variables turned out to be equally affected. For paced left atria an IC50 (causing 50% depression of contractile force) of 35 microM was determined. Right atria stopped beating at concentrations above 50 microM, thus hampering IC50 determination. The results indicate that anthracyclines exert an effect not related to receptor integrity, but directly to the functionality of heart muscle. To check whether radical stress can be involved in the observed negative inotropic effect, incubations with xanthine/xanthine oxidase (to produce reactive oxygen species) were performed. A pronounced negative effect on mouse atrial contraction was indeed observed. However, initially a positive inotropic effect accompanied by an increased resting tension were seen. It can be concluded that mouse atrium can be used as a model to compare anthracyclines and their metabolites with regard to their acute cardiotoxic effects.

Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation.

1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats upon an i.p. dose of 150 mumole/kg. At doses higher than 375 mumole/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, appeared to be slightly nephrotoxic in Wistar rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metabolism of DCDFE, probably involving epoxidation by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of two different mechanisms of bioactivation.

Laparoscopic diagnosis of mesenteric venous thrombosis.

Diagnosis of mesenteric venous thrombosis is almost always delayed, due to the aspecificity of the complaints and the clinical findings, as well as the laboratory investigations. Earlier diagnosis is essential to improve the present grim mortality rate. We report a case of mesenteric venous thrombosis in a 25-year-old female. Early diagnosis was made by gynecological laparoscopy. After resection and anastomosis, the outcome was uneventful.