Mustafa Birincioğlu

This prospective controlled follow-up study was designed to examine the effects of adnexal torsion on long-term ovarian histology and radical scavenger (FRS) activity, and subsequent viability following the detorsion of twisted ischaemic adnexa, in a primate centre of a university clinic. Adnexal torsion/occlusion was created by twisting the adnexa three times and fixing on to the side wall or by applying vascular clips in cycling female rats at 70 days of age. Following an ischaemic period of 4 to 36 h, the twisted adnexas were surgically removed and fixed. In the second group of rats, following the above ischaemic periods, the torsion/occlusion were relieved by detwisting or removing the vascular clips. Then the animals were reperfused for a week and adnexas were extirpated. After both ischaemia and reperfusion, the removed adnexas were examined histologically and tissue concentrations of glutathione peroxidase, superoxide dismutase, catalase and glutathione were determined. Regardless of the ischaemia time, all the twisted adnexas were black-bluish in appearance. Despite the gross ischaemic-haemorrhagic features, histological sections revealed negligible changes, with intact ovarian structure similar to controls in 4-24 h groups. Though decreased compared with controls, the change in tissue concentrations of FRS was not significant in 4-24 h groups. Only the 36 h group showed prominent congestion on all sections and a significant decrease in all radical scavenger concentrations studied. While no long-term reperfusion injury was observed histologically in 4-24 h groups, the 36 h group ended with adnexal necrosis. Our findings support the importance of early diagnosis and conservative surgical management (detorsion) in adnexal torsion. Lack of histological changes and unimpaired FRS metabolism are consistent with the recent data that vascular compromise is caused by venous or lymphatic stasis in early torsion and that adnexal integrity is not correlated with gross ischaemic appearance, thus providing evidence of adnexal resistance against ischaemia.

Both mitochondrial ATP-sensitive K+ (KATP) channels and the actin cytoskeleton have been proposed to be end-effectors in ischemic preconditioning (PC). For evaluation of the participation of these proposed end effectors, rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. PC by 5-min ischemia + 10-min reperfusion reduced infarct size by 60%. Diazoxide, a mitochondrial KATP-channel opener, administered before ischemia was protective. Protection was lost when diazoxide was given after onset of ischemia. Anisomycin, a p38/JNK activator, reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-hydroxydecanoate (5-HD), an inhibitor of mitochondrial KATP channels. Isolated adult rabbit cardiomyocytes were subjected to simulated ischemia by centrifuging the cells into an oxygen-free pellet for 3 h. PC was induced by prior pelleting for 10 min followed by resuspension for 15 min. Osmotic fragility was assessed by adding cells to hypotonic (85 mosmol) Trypan blue. PC delayed the progressive increase in fragility seen in non-PC cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5-HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data support a role for both mitochondrial KATP channels and cytoskeletal actin in protection by PC.

Tirapazamine is a bioreductively activated DNA-damaging agent that selectively kills the hypoxic cells found in solid tumors. This compound shows clinical promise and is currently being examined in a variety of clinical trials, including several phase III studies. It is well established that DNA is an important cellular target for tirapazamine; however, the structural nature of the DNA damage inflicted by this drug remains poorly understood. As part of an effort to understand the chemical events responsible for the hypoxia-selective cytotoxicity of this drug, the studies reported here are designed to characterize tirapazamine-mediated damage to the genetic information stored in the heterocyclic base residues of double-stranded DNA. Here, we used gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry to characterize and quantify oxidative DNA base damage mediated by tirapazamine. A multiplicity of modified bases including 8,5'-cyclopurine-2'-deoxynucleoside tandem lesions were identified and quantified. The results provide the first detailed insight regarding the structural identity of the DNA base lesions caused by this drug. Interestingly, it appears that the hypoxic conditions under which tirapazamine operates, along with the unique chemical properties of the drug, yield a unique variety of DNA base damage that is dominated by formamidopyrimidine and 5-hydroxy-6-hydropyrimidine lesions. Importantly, the results suggest that tirapazamine may generate a set of poorly repaired, potentially cytotoxic DNA base lesions that block DNA transcription and replication. Overall, the results indicate that DNA base damage may contribute to the biological effects of tirapazamine in vivo.

A functional homologue of human DNA glycosylase NEIL1 (hNEIL1) in mouse has recently been cloned, isolated, characterized, and named mouse NEIL1 (mNEIL1). This enzyme exhibited specificity for excision of oxidatively modified pyrimidine bases such as thymine glycol, 5,6-dihydrouracil, and 5-hydroxypyrimidines, using oligonucleotides with a single base lesion incorporated at a specific site. It also acted upon AP sites; however, no significant excision of 8-hydroxyguanine was observed [Rosenquist, T. A., Zaika, E., Fernandes, A. S., Zharkov, D. O., Miller, H., and Grollman, A. P. (2003) DNA Repair 2, 581-591]. We investigated the substrate specificity and excision kinetics of mNEIL1 for excision of oxidatively modified bases from high-molecular weight DNA with multiple lesions, which were generated by exposure of DNA in aqueous solution to ionizing radiation. Among a large number of pyrimidine- and purine-derived lesions detected and quantified in DNA, only purine-derived lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine were significantly excised. This finding establishes that mNEIL1 and its functional homologue hNEIL1 possess common substrates, namely, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine. Measurement of excision kinetics showed that mNEIL1 possesses equal specificity for these two formamidopyrimidines. This enzyme also excised thymine-derived lesions thymine glycol and 5-hydroxy-5-methylhydantoin, albeit at a much lower rate. A comparison of the specificity and excision kinetics of mNEIL1 with other DNA glycosylases shows that this enzyme is as efficient as those DNA glycosylases, which specifically remove the formamidopyrimidines from DNA.