Cell-autonomous requirement for DNaseII in nonapoptotic cell death.

DNA fragmentation is a critical component of apoptosis but it has not been characterized in nonapoptotic forms of cell death, such as necrosis and autophagic cell death. In mammalian apoptosis, caspase-activated DNase cleaves DNA into nucleosomal fragments in dying cells, and subsequently DNase II, an acid nuclease, completes the DNA degradation but acts non-cell autonomously within lysosomes of engulfing cells. Here we examine the requirement for DNases during two examples of programmed cell death (PCD) that occurs in the Drosophila melanogaster ovary, starvation-induced death of mid-stage egg chambers and developmental nurse cell death in late oogenesis. Surprisingly, we found that DNaseII was required cell autonomously in nurse cells during developmental PCD, indicating that it acts within dying cells. Dying nurse cells contain autophagosomes, indicating that autophagy may contribute to these forms of PCD. Furthermore, we provide evidence that developmental nurse cell PCD in late oogenesis shows hallmarks of necrosis. These findings indicate that DNaseII can act cell autonomously to degrade DNA during nonapoptotic cell death.

Determination of two mercapturic acids related to crotonaldehyde in human urine: influence of smoking.

Crotonaldehyde, an alpha,beta-unsaturated aldehyde, and a potent alkylating agent, is present in many foods and beverages, ambient air and tobacco smoke. A previous study indicated that two metabolites, 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) and 2-carboxyl-l-methylethylmercapturic acid (CMEMA), were excreted in rat urine after subcutaneous injection of crotonaldehyde. Herein, we report the development of a method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and deuterated analytes as internal standards, for the determination of HMPMA and CMEMA in human urine. The limits of quantification of the method were 92 and 104 ng/mL for HMPMA and CMEMA, respectively. The calibration curves for both compounds were linear up to 7500 ng/mL with R2 >0.99. It was found that cigarette smokers excreted about three to five-fold more HMPMA, and only slightly elevated amounts of CMEMA, in their urine compared to nonsmokers. In smokers, we also found significant correlations between the urinary excretion levels of HMPMA (but not CMEMA) and several markers of exposure for smoking, including the daily cigarette consumption, carbon monoxide in exhaled breath, salivary cotinine, and nicotine plus five of its major metabolites in urine. Smoking cessation or switching from smoking conventional cigarettes to experimental cigarettes with lower crotonaldehyde delivery led to significant reductions of urinary HMPMA excretion, but not CMEMA excretion. Alcohol consumption did not influence either urinary HMPMA or CMEMA excretion. We conclude that HMPMA is a potentially useful biomarker for smoking-related exposure to crotonaldehyde.