Role of metabolism and oxidation-reduction cycling in the cytotoxicity of antitumor quinoneimines and quinonediimines

Quinone(di)imines are nitrogen analogues of quinones in which one or both quinone oxygens are replaced by an imino group. A series of quinone(di)imines with antitumor activity has been studied for its in vitro chemical reactivity, metabolism, acute toxicity to primary cultured rat hepatocytes, and growth-inhibitory activity with Chinese hamster ovary (CHO) cells. The quinone(di)imines exhibited a wide range of activity as substrates for metabolism by hepatic microsomal flavoenzymes. The maximum rate of quinone(di)imine metabolism was more than 7.5-fold greater than reported for metabolism of quinones. Some quinone(di)imines formed free radicals that could be detected by electron spin resonance spectroscopy. 2-Amino-1,4-naphthoquinoneimine gave a short-lived electron spin resonance signal that could be detected only under aerobic conditions. 2,3',6-Trichloroindophenol gave an electron spin resonance signal in air that was stable for 24 h. Most quinone(di)imines underwent oxidation-reduction cycling to form the superoxide anion radical, but some quinone(di)imines, although rapidly metabolized, formed little or no superoxide anion radical. Quinone(di)imines were relatively toxic to hepatocytes and CHO cells, and some quinone(di)imines were more toxic to one cell type than the other. The log 1-octanol/water partition coefficient showed an optimal value of 2.61 for toxicity against both cell types. In hepatocytes the more toxic quinone(di)imines were the most rapidly metabolized. For a subgroup of quinone(di)imines toxicity to hepatocytes and CHO cells appeared to be related to the ability to form a semiquinone(di)imine free radical. Toxicity of quinone(di)imines to hepatocytes and CHO cells was not related to superoxide anion radical formation, and toxicity to CHO cells was not affected by exclusion of oxygen during exposure of the cells to the compounds. The rate of chemical addition of quinone(di)imines to reduced glutathione did not correlate with toxicity. An understanding of the mechanisms of acute toxicity and growth-inhibitory activity of quinone(di)imines could lead to the design of more selective quinonoid antitumor agents.     

Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism-based inhibition of thioredoxin reductase by antitumor quinoid compounds.

Quinoids undergo metabolism by a number of flavoenzymes. Reactive species formed during the metabolism of some quinoids might be anticipated to inhibit flavoenzyme activity. Several quinoids have been tested for their ability to inhibit rat liver thioredoxin reductase (TR). The antitumor quinones diaziquone and doxorubicin, and the quinoneimine 2,6-dichloroindophenol, were found to be inhibitors of the reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) by TR. The inhibition was most marked after incubation of the quinoid with NADPH and the enzyme for 60 min before adding DTNB, with Ki values of 0.5 microM for diaziquone, 0.5 microM for doxorubicin, and 0.07 microM for 2,6-dichloroindophenol. The three quinoids all produced a time-dependent and first order loss of TR activity. There was formation of electron spin resonance-detectable semiquinoid free radicals upon incubation of diaziquone, doxorubicin and 2,6-dichloroindophenol with TR and NADPH under anaerobic conditions. Oxygen radicals formed by redox cycling of the quinoids did not make a major contribution to the inhibition of TR by the quinoids, as shown by the absence of significant reversal of the inhibition by anaerobic incubation conditions and the lack of effect of the oxygen radical scavengers dimethyl sulfoxide, superoxide dismutase and catalase. It was not possible to demonstrate NADPH-dependent covalent binding of radiolabeled diaziquone or doxorubicin to the TR apoprotein. It is possible that the quinoids bind noncovalently to the enzyme apoprotein, or bind to the FAD prosthetic group. The results of the study suggest that some antitumor quinoids are mechanism-based inhibitors of TR showing metabolism- and time-dependent irreversible inhibition of enzyme activity.     

Plasma Concentrations of Mycophenolic Acid Acyl Glucuronide Are Not Associated with Diarrhea in Renal Transplant Recipients

The aim of this study was to determine whether plasma concentrations of the acyl (AcMPAG) and phenolic (MPAG) glucuronide metabolites of mycophenolic acid (MPA) were related to diarrhoea in renal transplant patients on mycophenolate mofetil (MMF) with cyclosporine (CsA) or tacrolimus (TCL). Blood samples (0, 30, 120 min) were taken at days 3, 10, week 4, months 3, 6 and 12 for determination of MPA, MPAG and AcMPAG. MPA-AUC was estimated using validated algorithms. Two hour AUCs were calculated for MPAG and AcMPAG. Immunosuppressive therapy consisted of CsA/MMF (n= 110) and of TCL/MMF (n= 180). In 70/290 (24%) patients 86 episodes of diarrhoea were recorded during 12 months. Significantly more patients on TCL (31.1%) suffered from diarrhea compared to CsA (12.7%). MMF dose, MPA-AUC and the 2 h AUCs of MPAG and AcMPAG did not differ between patients with and without diarrhoea. Plasma AcMPAG and MPAG concentrations were substantially higher in patients on CsA compared with TCL, while MPA-AUC was lower in the former group. These data support the concept that CsA inhibits the biliary excretion of MPAG and AcMPAG, thereby potentially reducing the risk of intestinal injury through enterohepatic recycling of MPA and its metabolites.     

Term delivery in a woman with severe congenital neutropenia, treated with growth colony stimulating factor

The patient was diagnosed in childhood as having severe congenital neutropenia and had recurrent admissions with severe infections. In 1987, prior to getting married, she was sterilized. She continued to require i.v. antibiotics when she contracted a severe infection. On one occasion, she was treated with growth colony stimulating factor (G- CSF). Her increased neutrophil count was sustained following this treatment. In June 1993, she wished to start a family and underwent in- vitro fertilization (IVF) treatment. G-CSF was given prior to oocyte retrieval. She conceived on her first cycle and an ultrasound scan revealed a singleton pregnancy. Throughout the course of the pregnancy, her white cell count was monitored closely and remained at <1.0x10(9)/l. The pregnancy progressed uneventfully and at 37 weeks gestation she was admitted for G-CSF injections. At 38 weeks she was delivered of a boy weighing 3350 g, by elective Caesarean section. His white cell count was normal. This is the first case of G-CSF being used before conception and during pregnancy in a patient with congenital neutropenia. It shows that advances in cytokine therapy and close interdisciplinary liaison can lead to a successful outcome and help patients, who would otherwise remain childless, to achieve a family.      

Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure [published erratum appears in Hum Reprod 1998 Mar;13(3):780]

The aim of the study was to determine whether a prior diagnostic testicle biopsy can predict success or failure of testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) in patients with non-obstructive azoospermia caused by testicular failure, and what is the minimum threshold of sperm production in the testis which must be surpassed for spermatozoa to reach the ejaculate. Forty- five patients with non-obstructive azoospermia caused by testicular failure underwent diagnostic testicle biopsy prior to a planned future TESE-ICSI procedure. The diagnostic testicle biopsy was analysed quantitatively, and correlated with the quantitative findings of spermatogenesis in patients with normal spermatogenesis, as well as with the results of subsequent attempts at TESE-ICSI. Men with non- obstructive azoospermia caused by germinal failure had a mean of 0-6 mature spermatids/seminiferous tubule seen on a diagnostic testicle biopsy, compared to 17-35 mature spermatids/tubule in men with normal spermatogenesis and obstructive azoospermia. These findings were the same for all types of testicular failure whether Sertoli cell only, maturation arrest, cryptorchidism, or post-chemotherapy azoospermia. Twenty-two of 26 men with mature spermatids found in the prior testis biopsy had successful retrieval of spermatozoa for ICSI, 12 of their partners became pregnant, and are either ongoing or delivered. The study suggests that 4-6 mature spermatids/tubule must be present in the testis biopsy for any spermatozoa to reach the ejaculate. More than half of azoospermic patients with germinal failure have minute foci of spermatogenesis which are insufficient to produce spermatozoa in the ejaculate. Prior diagnostic testicle biopsy analysed quantitatively (for the presence of mature spermatids) can predict subsequent success or failure with TESE-ICSI. Incomplete testicular failure may involve a sparse multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a regional distribution. Therefore, it is possible that massive testicular sampling from many different regions of the testes may not be necessary for successful TESE-ICSI.