Hypochlorous acid/N-chloramines are naturally produced DNA repair inhibitors.

Human mononuclear leukocytes (HML) respond to oxidative DNA damage by activation of ADP ribosylation and initiation of DNA repair synthesis (i.e. unscheduled DNA synthesis, UDS), whereas neutrophils do not. When neutrophils are added to HML cultures in ratios up to 4:1 ADP ribosylation becomes inhibited to approximately 50-60%. The ability of neutrophils to inhibit HML ADP ribosylation was shown to be dependent on H2O2, chloride ions and myeloperoxidase, which in turn are factors known to govern HOCl and N-chloramine production by phagocytic cells. HOCl and a model N-chloramine, chloramine T, were shown to give a dose-dependent inhibition of DNA repair using four independent estimates, namely ADP ribosylation, UDS and the repair of DNA strand breaks estimated by nucleoid sedimentation and alkaline elution profiles. All the DNA repair measurements used on HML were inhibited approximately 70-80% by 100 microM doses of HOCl or chloramine T, which was considered a biologically relevant dose because: (i) viable neutrophils equal in concentration to those found in blood could easily produce 100 microM levels in short-term culture; (ii) 100 microM doses of these agents were not acutely cytotoxic judged by trypan blue stained cells after 30-60 min exposure and under the conditions used for assay, but yet they abolished 86-95% of the growth response of HML to phytohemagglutinin.

Reduced nonspecific steroidal esterase activity in human malignant tumor tissue from liver, colon and breast when compared to peritumoral and normal tissue levels.

The levels of a nonspecific steroidal esterase estimated as alpha naphthyl acetate esterase (ANAE) activity were measured in apparently normal (peritumoral) tissue of liver, colon and breast cancer patients, in malignant tumor tissue from the same patients and in normal colon tissue from healthy individuals who never had colon cancer. Cancer tissue from all three organs showed 35-79% reduced levels of ANAE activity when compared to peritumoral tissue of the same organ (p < 0.03). When ANAE activity of normal colon tissue from healthy individuals who never had colon cancer was compared with peritumoral colon tissue from colon cancer patients, which were combined with colonic biopsies from cancer-free patients previously operated on for colon cancer, a 50% reduced ANAE activity was observed in these tissues compared to the normal (p < 0.005). A highly significant reduction of enzyme activity was also found when malignant colon tissue from the cancer patients was compared to normal colon tissue from healthy individuals who never had colon cancer.

Non-specific steroidal esterase activity and distribution in human and other mammalian tissues.

An NADPH dependent arylamine carcinogen and fatty acid steroid ester metabolizing esterase activity belonging to the B- or carboxylesterase class of non-specific esterase (EC 3.1.1.1) was measured by two different methods: (i) a spectrophotometric assay using alpha naphthyl acetate (ANA) as substrate and (ii) a radiometric method using the conversion of beclomethasone-17,21-dipropionate to beclomethasone-17-monopropionate as the endpoint. The two methods were strongly correlated when assayed in human mononuclear leukocytes (r = 0.89, P < 0.0001) and human mammary tissue (r = 0.91, P < 0.0001). Hence it was concluded that the two substrates are metabolized at least in part by the same enzyme. This esterase activity was abundant in human monocytes, present in T-lymphocytes and equally divided between CD4 and CD8 T-lymphocyte subsets. The same activity was expressed in human liver, colon, stomach, breast and brain tissues. The distribution of this esterase in human tissues showed high activity in liver, intermediate activity in colon, stomach and breast and low activity in brain tissue. The interorgan distribution observed in human tissues was closely mimicked when the esterase activity was assessed in liver, colon and brain tissues from three mouse strains and three rat strains. The non-specific steroidal esterase activity determined by ANA metabolism in human mammary tissue was shown to be reproducible when assayed as triplicate samples from each of 16 different women (intraclass correlation coefficient 67.3%, P < 0.03). The interindividual variation in mammary tissue was high (18.4-fold) and there was a positive correlation between the esterase activity and age (r = 0.58, P < 0.01), as well as a tendency toward bimodal distribution. To our knowledge, these data represent the first systematic study of interorgan and interspecies comparisons of a non-specific steroidal esterase activity.

Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH.

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using alpha-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.

Adenosine diphosphate ribosyl transferase responses to a standardized dose of hydrogen peroxide in the mononuclear leukocytes of patients with a diagnosis of cancer.

Mononuclear leukocytes from 151 patients with cancer of various organs and from 467 apparently cancer-free individuals were exposed, in vitro, to H2O2 (100 microM) and the effects of the exposure on the activity of adenosine diphosphate ribosyl transferase (ADPRT) were determined. First, the reproducibility of this test procedure was established as satisfactory, by comparing the results of assays performed independently by two investigators, and by measuring ADPRT in cells from two individuals over a 9-week period. The test data were analyzed by multiple linear regression, and the correlation of cancer diagnosis, age, sex and smoking habits with ADPRT values was determined. The strongest correlate was cancer diagnosis. We considered categorizing ADPRT values as high and low, with a cut-off value that would substantially distinguish cancer from cancer-free individuals. When a cut-off value of 1200 c.p.m. TCA ppt [3H]NAD+/5 x 10(5) cells was applied to the complete test material, it was found that ADPRT values from cancer patients were more frequently below the cut-off than values from disease-free individuals: the relative risk estimate (odds ratio) was 13.8. When a similar analysis was done on values from lung cancer patients and smoking disease-free individuals, the odds ratio was 73.5. However, a cut-off value of 2000 c.p.m. TCA ppt [3H]NAD+/5 x 10(5) cells was most effective in distinguishing lung cancer patients (the largest cancer group, n = 96) from smoking non-cancer individuals: that value provided better sensitivity (85%) and specificity (81%) than other cut-off values tested in the range 1200-2000 c.p.m. Further, in the case of lung cancer, possible effects of anatomical site, and of staging and pathology on ADPRT values was analyzed by the chi-squared test: no significant associations were found. These data support the value of the ADPRT test in detecting early stage lung cancer regardless of location or pathological type.

The nonspecific esterases of human mononuclear leukocytes metabolize arylamine carcinogens and steroids esters.

The presence of non-specific esterases in various leukocyte subfractions of whole blood is well established, but no endogenous substrates or function for these esterases have been identified. Here we report on the metabolism of N-acetoxy-2-acetylaminofluorene (NA-AAF) and beclomethasone-17-21-dipropinate (BDP) in viable human mononuclear leukocytes (HML). Conversion of NA-AAF to DNA binding intermediates and BDP to beclomethasone-17-monopropionate by a common esterase was demonstrated and then further characterized by a broad spectrum of effectors including well-established inhibitors and substrates for the nonspecific esterases. Two esters, beta estradiol-17-propionate and alpha naphtyl propionate, competitively inhibited this esterase activity. Together, these data identify at least one isozyme of A- or B-classes of HML nonspecific esterases as being responsible for the metabolism of NA-AAF and BDP. That HML nonspecific esterases may be functionally involved in arylamine carcinogenes (i.e. as it may relate to immune function) and in the endogenous production of steroids from their naturally occurring esters emphasizes the importance of continuing their characterization.

DNA repair synthesis in individuals with and without a family history of cancer.

The influence of family history on DNA repair synthesis, unscheduled DNA synthesis (UDS), was assessed in volunteers with or without a family history of cancer. UDS, following treatment of mononuclear leukocytes with N-acetoxy-2-acetylaminofluorene, was measured as the incorporation of [3H]thymidine into DNA in the presence of hydroxyurea. The positive family history group (n = 71) had an average of 2.4 first-degree relatives with cancer, defined as any major cancer, excluding skin cancer: 31 participants reported that cancer occurred in both their parents. The "no family history' comparison group (n = 29) had no family history of cancer through the second degree. There was a significant reduction in UDS in cells from individuals with family history, compared to those with no family history (P greater than 0.002). This relationship was not explained by factors known to influence UDS, such as age, smoking or hypertension. We conclude that reduced UDS in mononuclear leukocytes is associated with a family history of any major cancer, and is not confined to a history of cancer of any single organ site. This conclusion is further supported by the observation that individuals (n = 13) with parents who had an earlier onset of cancer (less than 60 years) also had a significantly lower DNA repair synthesis than those (n = 18) whose parents had later diagnosis of cancer (greater than 60 years).

Relationship between the covalent binding of N-acetoxy-2-acetylaminofluorene to DNA and a steroidal esterase activity in human mononuclear leukocytes.

A method for the quantitative assessment of steroidal esterase activity in viable human mononuclear leukocytes (HML) has been developed. It is based on estimating the conversion of [3H]beclomethasone-17,21-dipropionate (BDP) to beclomethasone-17-monopropionate (BMP) using TLC on silica gel 60 F-254 plates developed in a solvent system of chloroform/methanol (97:3, v/v). The cell assay procedure was dependent on BDP concentration, incubation time and cell concentration. The steroidal esterase activity was completed for by N-acetoxy-N-acetyl-2-aminofluorene (NA-AAF) and completely inhibited by 100 microM paraoxon. When [3H]NA-AAF binding to DNA was used as an indicator of HML esterase (deacylase) activity, BDP functioned as a substrate inhibitor. Parallel estimations of BDP metabolism and NA-AAF binding to DNA indicated striking correlations in the interindividual variations (r = 0.62, P less than 0.001) and in relation to the menstrual cycle events of a healthy female. Hence, these data indicate that both BDP and NA-AAF are metabolized by the same non-specific steroidal esterase present in HML.