Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6- alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector- transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane- derived species to alkylate Trx.      

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp- like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.      

Cytogenetic and molecular genetic analysis of tumorigenic human bronchial epithelial cells induced by radon alpha particles

To establish a cell culture model for lung carcinogenesis, independent populations of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were treated with high linear energy transfer radon-simulated alpha-particles, expanded and xenotransplanted into Nu/Nu mice. Six independent cell lines were established from tumors that developed from three separate radiation treatments as follows: treatment (Tx) 1 (30 cGy--two doses), H2BT, Tx 2 (30 cGy-- single dose), R30T1L, R30T2 and R30T3L, Tx 3 (30 cGy--single dose), H1ATN and H1ATBA1. Cytogenetic analysis revealed common changes in all tumor lines: loss of the Y chromosome (ch), one of three copies of ch8, one of three copies of ch14, and one of two copies of ch4p16-pter and ch11p15-pter. Analysis of polymerase chain reaction-amplified short tandem repeats of informative loci confirmed the loss of chY in all lines and loss of heterozygosity (LOH) at eight loci spanning the length of ch8 in all lines from Tx's 1 and 2. Our data support previous studies indicating the presence of tumor suppressor genes on ch8. LOH also was confirmed on ch14 at locus D14S306 in all cell lines from Tx 2 and in one of two lines from Tx 3. This region, 14q12-q13, may contain changes in one of the five known somatostatin receptor genes (SSTR1). No LOH was detected at any of the informative loci tested for on ch4 or ch11.      

The influence of reported paternal attitudes on the decision to breast-feed

Objective: To identify factors that influence a woman's decision to breast-feed.
Methodology: Five hundred and fifty-six women were recruited from the maternity wards of two Perth hospitals. Data were collected from a self-administered questionnaire completed by participants prior to discharge. Logistic regression analysis was used to determine factors influencing the initiation of breast-feeding.
Results: At discharge from hospital 83.8% of women were breast-feeding, including 6% who were giving complementary formula feeds. After controlling for potentially confounding demographic and biomedical factors, the father's reported preference for breast-feeding was found to be the most important factor influencing a woman's decision to breast-feed (OR 10.18).
Conclusion: Fathers participate in and influence the choice of infant feeding method and should be included in breast-feeding discussions.     

Variability of results of cavernous nerve stimulation during radical prostatectomy

PURPOSE: We assess the reliability of intraoperative cavernous nerve stimulation for producing an erectile response during radical prostatectomy. MATERIALS AND METHODS: In 61 patients cavernous nerve function was assessed during radical retropubic prostatectomy using a CaverMap nerve stimulator. Control stimulation was also performed before and after prostatic dissection by placing the nerve stimulator tip on the anterior bladder wall. An increase in penile circumference measured by the device was considered a tumescence response while any measurable detumescence was also categorized. RESULTS: Patient age ranged 43 to 72 years (mean 59.8). Before apical dissection 41% and 46% had tumescence, 31% and 21% had detumescence, and 28% and 33% had no response with stimulation of the neurovascular bundle and anterior bladder wall, respectively. After dissection 42% and 25% had tumescence, 16% and 18% had detumescence, and 42% and 57% had no response with stimulation of the neurovascular bundle and anterior bladder wall, respectively. CONCLUSIONS: A response to neurovascular bundle stimulation using this device does not necessarily correlate with the precise anatomical location of the cavernous nerves. There is considerable background variability related to anesthesia, surgical manipulation and other undefined factors that may cause minor but measurable changes in penile circumference.     

Genetic prothrombotic risk factors in women with unexplained pregnancy loss

INTRODUCTION: Inherited thrombophilia has been associated with unexplained recurrent pregnancy loss (RPL) and stillbirth. This thrombotic tendency can manifest as thrombotic lesions in the placenta, and may lead to abortion and stillbirth. The aim of our case-control study was to investigate the prevalence of FVL and FII G20210A in women with adverse pregnancy outcome, compared to the prevalence of the same mutations in our health control group. MATERIALS AND METHODS: 102 consecutive women with unexplained pregnancy loss (55 with history of RPL, and 47 with history of stillbirth) were studied for hereditary thrombophilia. The health control group consisted of 217 healthy women from the general population. RESULTS AND CONCLUSIONS: Of the 55 women with recurrent abortions, we found the same prevalence for the FVL and the FII G20210A(9.1%, 5 pts). (p=NS compared to control group). Of the 47 women with stillbirth, 11 (23.4%) had the FVL and 9 (19.1%) had the FII G20210A(p<0.0005 for both mutations). In our experience the prevalence of FVL and the FII G20210Amutations was significantly higher in women with unexplained stillbirth, instead the prevalence of genetic thrombophilia was high but not statistically significant in women with recurrent pregnancy loss.