Association between latent toxoplasmosis and cognition in adults: a cross-sectional study.

Latent infection from Toxoplasma gondii (T. gondii) is widespread worldwide and has been associated with cognitive deficits in some but not all animal models and in humans. We tested the hypothesis that latent toxoplasmosis is associated with decreased cognitive function in a large cross-sectional dataset, the National Health and Nutrition Examination Survey (NHANES). There were 4178 participants aged 20-59 years, of whom 19.1% had IgG antibodies against T. gondii. Two ordinary least squares (OLS) regression models adjusted for the NHANES complex sampling design and weighted to represent the US population were estimated for simple reaction time, processing speed and short-term memory or attention. The first model included only main effects of latent toxoplasmosis and demographic control variables, and the second added interaction terms between latent toxoplasmosis and the poverty-to-income ratio (PIR), educational attainment and race-ethnicity. We also used multivariate models to assess all three cognitive outcomes in the same model. Although the models evaluating main effects only demonstrated no association between latent toxoplasmosis and the cognitive outcomes, significant interactions between latent toxoplasmosis and the PIR, between latent toxoplasmosis and educational attainment, and between latent toxoplasmosis and race-ethnicity indicated that latent toxoplasmosis may adversely affect cognitive function in certain groups.

Intermittent Palm Cooling's Impact on Resistive Exercise Performance.

To examine palm cooling's (15 °C) impact, subjects performed 3 four-set leg press workouts in a randomized sequence. Per workout they received 1 of 3 treatments: no palm cooling, palm cooling between sets, or palm cooling between sets and post-exercise. Dependent variables were examined with three-way ANOVAs; average power underwent a three-way ANCOVA with body fat percentage as the covariate. Simple effects analysis was our post hoc and α=0.05. Left hand skin temperatures produced a two-way interaction (no palm cooling, palm cooling between sets>palm cooling between sets and post-exercise at several time points). A "high responder" subset had their data analyzed with an additional three-way ANOVA that again produced a two-way interaction (palm cooling between sets>no palm cooling>palm cooling between sets and post-exercise at multiple time points). Blood lactate results included a two-way interaction (no palm cooling>palm cooling between sets, palm cooling between sets and post-exercise at 0 min post-exercise). Average power yielded a two-way interaction (palm cooling between sets, palm cooling between sets>no palm cooling for the fourth set). Intermittent palm cooling hastened heat removal and blood lactate clearance, as well as delayed average power decrements.

Randomized clinical trial of pigtail catheter versus chest tube in injured patients with uncomplicated traumatic pneumothorax.

BACKGROUND: Small pigtail catheters appear to work as well as the traditional large-bore chest tubes in patients with traumatic pneumothorax, but it is not known whether the smaller pigtail catheters are associated with less tube-site pain. This study was conducted to compare tube-site pain following pigtail catheter or chest tube insertion in patients with uncomplicated traumatic pneumothorax. METHODS: This prospective randomized trial compared 14-Fr pigtail catheters and 28-Fr chest tubes in patients with traumatic pneumothorax presenting to a level I trauma centre from July 2010 to February 2012. Patients who required emergency tube placement, those who refused and those who could not respond to pain assessment were excluded. Primary outcomes were tube-site pain, as assessed by a numerical rating scale, and total pain medication use. Secondary outcomes included the success rate of pneumothorax resolution and insertion-related complications. RESULTS: Forty patients were enrolled. Baseline characteristics of 20 patients in the pigtail catheter group were similar to those of 20 patients in the chest tube group. No patient had a flail chest or haemothorax. Pain scores related to chest wall trauma were similar in the two groups. Patients with a pigtail catheter had significantly lower mean(s.d.) tube-site pain scores than those with a chest tube, at baseline after tube insertion (3.2(0.6) versus 7.7(0.6); P < 0.001), on day 1 (1.9(0.5) versus 6.2(0.7); P < 0.001) and day 2 (2.1(1.1) versus 5.5(1.0); P = 0.040). The decreased use of pain medication associated with pigtail catheter was not significantly different. The duration of tube insertion, success rate and insertion-related complications were all similar in the two groups. CONCLUSION: For patients with a simple, uncomplicated traumatic pneumothorax, use of a 14-Fr pigtail catheter is associated with reduced pain at the site of insertion, with no other clinically important differences noted compared with chest tubes. REGISTRATION NUMBER: NCT01537289 (http://clinicaltrials.gov).

Diabetic education in rural areas.

INTRODUCTION: Diabetes mellitus type II is a growing concern in the USA, with 6% of the population diagnosed with diabetes and another 5% having pre-diabetes. The prevalence of diabetes is 17% higher in rural areas than in central cities{1}. Adult diabetics living in rural areas often see negative outcomes related to their limited access to care, cultural barriers, and lack of educational resources. This article seeks to evaluate best evidence-based strategies directed at improving diabetic outcomes of rural populations through hemoglobin A1C (HbA1C) reductions. METHOD: A search of Medline, CIHNAL, PubMed, and Sage Pub was undertaken. The search was structured around the following key terms: adult, diabetes, education, hemoglobin a1c, and rural. The search limits were set to English-language publications between 2004 and 2012 in industrialized countries. Only articles from scholarly, peer-reviewed publications were considered. Literature that used an inpatient setting, focused on children or adolescents, and did not meet any inclusion criteria were excluded from this review. RESULTS: A total of 15 articles met the selection criteria from the 1819 citations sourced from the search. After reviewing the sources, nutritional patient education, motivational counseling and lifestyle modifications were found to be the most influential factors that favorably changed measurable outcomes for this population. Education for providers did not have an appreciable effect on patient outcomes. CONCLUSION: This review adds to the literature by outlining best-practice guidelines for evidence-based practice based on current research. Primary care providers in rural areas should encourage their patients to actively participate in diabetes education when possible, and provide this education in a culturally competent manner.

Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice.

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4(+) T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Exposure assessment and process sensitivity analysis of the contamination of Campylobacter in poultry products.

Studies were conducted in a Thai poultry plant to identify the factors that affected numbers of Campylobacter jejuni in chicken carcasses. The concentrations of Campylobacter were determined using the SimPlate most probable number and modified charcoal cefoperazone deoxycholate plating methods. Results indicated that the mean concentrations of C. jejuni in carcasses after scalding, plucking, and chilling were 2.93 ± 0.31, 2.98 ± 0.38, 2.88 ± 0.31, and 0.85 ± 0.95 log cfu, whereas the concentrations of C. jejuni in the scalding tank water, plucked feathers, and chicken breast portion were 1.39 ± 0.70, 3.28 ± 0.52, and 0.50 ± 1.22 log cfu, respectively. Sensitivity analysis using tornado order correlation analysis showed that risk parameters affecting the contamination of C. jejuni in the chicken slaughter and processing plant could be ranked as chilling water pH, number of pathogens in the scald tank water, scalding water temperature, number of C. jejuni on plucked feathers, and residual chlorine in the chill water, respectively. The exposure assessment and analysis of process parameters indicated that some of the current critical control points were not effective. The suggested interventions included preventing fecal contamination during transportation; increasing the scalding temperature, giving the scalding water a higher countercurrent flow rate; reducing contamination of feathers in the scalding tank to decrease C. jejuni in the scalding water; spraying water to reduce contamination at the plucking step; monitoring and maintaining the chill water pH at 6.0 to 6.5; and increasing the residual chlorine in the chill water. These interventions were recommended for inclusion in the hazard analysis and critical control point plan of the plant.

Antemortem inhibition of phosphoinositide 3-kinase causes changes in meat quality traits in broiler chickens.

Phosphoinositide 3-kinase (PI3K) is an enzyme that mediates endocrinological responses, such as intracellular signaling of insulin and growth factors, and plays important roles in muscle homeostasis and growth. In this study, the effect of antemortem PI3K activity on meat quality traits was investigated using broiler chickens whose PI3K was inhibited pharmacologically. Breast and thigh muscles were harvested from broilers treated with the PI3K inhibitor wortmannin, and meat quality traits were evaluated by determination of color, water-holding capacity, and breaking strength. The pH and concentrations of glycogen and free amino acids were also investigated as determinants of the chemical properties of meat. The results indicated that antemortem PI3K inhibition by wortmannin modified breast muscle color with lower L∗ values (P < 0.05) and b∗ values (P < 0.05) and higher a∗ values (P < 0.05). Antemortem PI3K inhibition also increased the water-holding capacity of breast muscles (P < 0.05), although breaking strength was not much affected. In addition, antemortem PI3K inhibition increased the concentrations of free amino acids in breast muscles, especially arginine (P < 0.05) and glutamic acid (P < 0.05). Similar effects were observed in thigh muscles. Lower glycogen levels at sacrifice (P < 0.05) and the resultant higher pH during the postmortem period (P < 0.05) were associated with PI3K inhibition-induced changes in meat quality traits. The wortmannin-treated muscles shared certain features with dark, firm, and dry meat which is a common abnormal meat. These findings suggest that antemortem PI3K activity contributes to meat quality traits and is involved in the molecular mechanism of the production of meat quality abnormalities.

Differential expression of eight defensin genes of N. benthamiana following biotic stress, wounding, ethylene, and benzothiadiazole treatments.

Eight Nicotiana benthamiana defensin genes were identified that could be divided into two classes with class II defensins being longer than class I defensins due to an additional acidic C-terminal domain. Class I defensins were NbDef1.1, NbDef1.2, NbDef1.3, NbDef1.4, NbDef1.5, and NbDef1.6, and class II were Nbdef2.1 and NbDef2.2. Relative RT-PCR showed that NbDef1.1, NbDef1.2, and NbDef1.4 had relatively similar expression levels in healthy leaves, stems, roots, flowers, and seeds. However, Nbdef1.3, NbDef1.5, and NbDef1.6 had varying degrees of tissue specific expression, and Nbdef2.1 and NbDef2.2 had strictly flower-specific expression. None of the defensins were significantly induced by infection by Colletotrichum destructivum or C. orbiculare. However, infection by Pseudomonas syringae pv. tabaci resulted in increased expression of Nbdef1.2 and Nbdef2.2, and decreased expression of NbDef1.1, NbDef1.4, and NbDef1.6. In the hypersensitive response of N. benthamiana containing Pto with P. syringae pv. tabaci containing AvrPto, only NbDef2.2 was significantly up-regulated. Expression of the genes was also affected by abiotic treatments. Both wounding and ethylene treatments resulted in a strong induction of NbDef2.2 and a moderate to weak induction of NbDef1.1, NbDef1.2, and NbDef1.4. Only weak or no induction was observed with treatment with benzothiadiazole. The expression of these eight defensin genes demonstrates that only a small fraction of the members of a defensin gene family will respond to a particular hemibiotrophic pathogen as well as to abiotic stress or signaling molecules.

Metastatic malignant melanoma.

Metastatic malignant melanoma is an incurable malignancy with extremely poor prognosis. Patients bearing this diagnosis face a median survival time of approximately 9 months with a probability of surviving 5 years after initial presentation at less than 5%. This is contrasted by the curative nature of surgical resection of early melanoma detected in the skin. To date, no systemic therapy has consistently and predictably impacted the overall survival of patients with metastatic melanoma. However, in recent years, a resurgence of innovative diagnostic and therapeutic developments have broadened our understanding of the natural history of melanoma and identified rational therapeutic targets/strategies that seem poised to significantly change the clinical outcomes in these patients. Herein we review the state-of-the-art in metastatic melanoma diagnostics and therapeutics with particular emphasis on multi-disciplinary clinical management.

Detection and partial characterization of an inhibitor of plasminogen activator in human platelets.

In this study, we demonstrate the presence of a previously undescribed fibrinolytic inhibitor in human serum. It has an apparent molecular weight of 50,000 and is not detected in serum derived from platelet-poor plasma, suggesting that it originates from platelets. This conclusion is supported by a number of observations. For example, extracts of washed, gel-filtered human platelets contain an inhibitor of similar activity and size, and physiological concentrations of thrombin induce its release from the platelets. Moreover, the kinetics and dose dependency of this release are similar to those observed for the release of platelet factor 4, and the release of both molecules is blocked by pretreating the platelets with prostaglandin E1 and theophylline. Mixing experiments, which were devised to investigate the specificity of the inhibitor, showed that the fibrinolytic activity initiated by both urokinase and tissue-type plasminogen activator was blocked by platelet releasate in a dose-dependent manner. In both cases, the amount of inhibition increased when the releasates were preincubated with the purified activators, indicating a direct interaction between the activators and an inhibitor(s). The inhibitory activity was removed by preincubating the releasates with antiserum prepared against an antiactivator purified from cultured bovine aortic endothelial cells. These results indicate that platelets contain an inhibitor which is released by thrombin, inhibits both urokinase and tissue-type plasminogen activator, and is immunologically similar to an inhibitor produced by endothelial cells. This molecule may represent a new class of inhibitors, the antiactivators, which function together with alpha 2-antiplasmin to regulate the fibrinolytic system of the blood. Its release from platelets by thrombin may protect the growing thrombus against premature dissolution initiated by plasminogen activators released by the endothelium.

Interrelationships of protein and DNA syntheses during replication of mammalian cells.

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase.

Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34+ expression. Nine subjects were infused with CD34+-enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34+ peripheral blood progenitor cells in the setting of chemotherapy.

A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase.

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O(6)-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.

Phase I study of streptozocin- and carmustine-sequenced administration in patients with advanced cancer.

BACKGROUND: Fewer than 20% of patients with nonhematologic malignancies treated with chloroethylnitrosoureas (CENUs) respond, but streptozocin (STZ), which depletes O6-methylguanine-DNA-methyltransferase (MGMT), has been shown to reverse resistance to CENUs in vitro. PURPOSE: The purpose of this phase I study was to determine (a) the maximum tolerated dose (MTD) of carmustine (BCNU), a CENU, plus a fixed dose of STZ; (b) the toxic effects of the drugs; and (c) the effects on peripheral blood mononuclear cells (PBMC). METHODS: A clinical phase I study of STZ followed by BCNU was designed to simulate conditions that produce maximal sensitization of CENU-resistant HT-29 cells in vitro. Patients received a 20-minute infusion of the MTD of STZ (2 g/m2) followed 1 hour later with a 60-minute infusion of BCNU (100, 125, 137.5, or 150 mg/m2). Treatment was repeated after 6 weeks. Twenty-four patients with advanced malignancies received 32 courses of therapy (range, 1-2 courses). RESULTS: The MTD of BCNU was 125 mg/m2. The dose-limiting toxic effect was thrombocytopenia occurring about 22 days after treatment, with recovery between days 28 and 35. Transient hypophosphatemia and proteinuria were common, and serum creatinine was elevated in 9% of the courses. Two patients who received therapy died--one due to pulmonary toxic effects and one due to hepatic toxic effects. Two patients with previously untreated carcinoid achieved partial response. In three patients, MGMT levels in PBMC were more than 85% depleted after STZ administration and more than 90% depleted after BCNU infusion. CONCLUSIONS: These results show that the magnitude of MGMT depletion by STZ in PBMC is in the range necessary to produce sensitivity to CENUs in resistant cell lines but also that, when BCNU is combined with STZ, the MTD of BCNU is about 50% that of BCNU as a single agent and that platelet count suppression occurs earlier. IMPLICATIONS: We plan to conduct phase II studies of STZ plus BCNU in tumor types with low response to CENUs. One of the major goals will be to demonstrate that depletion of MGMT is greater in tumor cells than in normal cells.

Changes in c-myc and c-fos expression in a human tumor cell line following exposure to bifunctional alkylating agents.

This study was initiated to determine if DNA-damaging chemotherapeutic agents can suppress the expression of oncogenes. The effects of three structurally related bifunctional alkylating agents on the steady state mRNA levels of c-myc, c-fos, N-ras, and beta-actin in the human colon carcinoma cell line Colo320HSR were examined. Colo320HSR has an amplified c-myc oncogene, which is highly overexpressed, and is assumed to be one of the transforming genes of this cell line. Two concentrations of mechlorethamine, L-phenylalanine mustard, and 4-hydroperoxycyclophosphamide, which produced 1 or 3 log cell kills were used to examine the effects of drug exposure on the expression of specific genes. Steady state mRNA levels were measured by Northern blot analysis. Following a 1-h drug exposure, RNA was isolated from cells at 0, 6, 12, and 24 h following drug removal. The agents used produced changes in the expression of specific genes, and all three did so in a similar fashion. Immediately following drug removal, the steady state expression of c-myc in treated cells was increased 2- to 3-fold compared to control. At 6 and 12 h following drug removal, c-myc levels were depressed 2.5- to 5-fold. By 24 h, c-myc expression approached, but remained below, control levels. Immediately following drug removal, c-fos levels were increased 3- to 4-fold, and from 6 to 24 h following drug removal, c-fos levels gradually return to, or fell below low basal levels. During the 24-h time course, drug treatment had little or no effect on the steady state levels of N-ras or beta-actin. These data support the hypothesis that alkylating agents may suppress the expression of specific transforming genes.

Extended depletion of O6-methylguanine-DNA methyltransferase activity following O6-benzyl-2'-deoxyguanosine or O6-benzylguanine combined with streptozotocin treatment enhances 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

We have recently suggested that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance might require complete inactivation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) for at least 24 h following BCNU administration (22). This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibition, with partial or complete recovery of MGMT activity by 24 h. We were able to modulate the recovery rate of MGMT activity following BG or dBG administration by repeated washing of cells with complete medium. Subsequent to equally inhibitory doses of BG (100 microM) or dBG (1.0 mM) treatment without washing, MGMT activity was completely inactivated for 24 h. However, MGMT activity recovered to control levels by 24 h when cells were treated with BG or dBG and washed 4 times with complete medium. This recovery was completely inhibited for 24 h by combining BG or dBG with 2.5 mM STZ. These differential repletion profiles produced disparate potentiation of BCNU cytotoxicity. The regimens which produced complete inactivation of MGMT for 24 h produced the greatest enhancement of BCNU cytotoxicity. BG or dBG (without a wash) potentiated BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via washing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergistic cell kill. The addition of STZ to BG or dBG inhibited this temporal recovery for 24 h and potentiated BCNU cytotoxicity by approximately 4 logs. These data further demonstrate that extended depletion of MGMT is required for optimal reversal of BCNU resistance. Because a three-drug combination of BG-STZ-BCNU or dBG-STZ-BCNU consistently produced greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that the design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importance of extended depletion of MGMT in order to increase the possibility of antitumor responses.

Inhibition of cis-diammine-1,1-cyclobutane dicarboxylatoplatinum(II)-induced DNA interstand cross-link removal and potentiation of cis-diammine-1,1-cyclobutane dicarboxylatoplatinum(II) cytotoxicity by hydroxyurea and 1-beta-D-arabinofuranosylcytosine.

1-beta-D-Arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) were investigated as potential DNA repair inhibitors with cis-diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA). HU plus ara-C, known inhibitors of DNA excision-repair, had previously been found to produce cytotoxic synergy and delayed removal of DNA interstrand cross-links with cis-diamminedichloroplatinum(II) (DDP). Since CBDCA and DDP share a common active intermediate, it should be possible to reproduce this interaction with CBDCA. However, the stable dicarboxylate chelate ring structure of CBDCA results in kinetics that differ significantly from those of DDP, due to slower hydrolysis to the active species. DNA adducts form more slowly, with interstand cross-links peaking approximately 12-h later and disappearing more gradually than in the case of DDP. It was therefore expected that a longer antimetabolite exposure might be required for repair inhibition with CBDCA. The 12-h exposure to HU plus ara-C previously found effective with DDP produced no cytotoxic synergy with a 2-h CBDCA exposure. Lengthening the antimetabolite treatment to 24 h resulted in approximately 1 log of synergistic toxicity, while a 24-h simultaneous exposure to HU, ara-C, and CBDCA resulted in 2 logs. Cells exposed to all three drugs showed a 2- to 3-fold greater level of interstrand cross-links after 36- to 48-h of incubation following drug removal, compared to CBDCA alone. Taken together, these findings suggest that HU plus ara-C modulates the repair of platinum-DNA adducts and establishes an effective in vitro schedule at clinically achievable concentrations for the use of those antimetabolites with CBDCA.

Measurements of DNA damage in Chinese hamster cells treated with equitoxic and equimutagenic doses of nitrosoureas.

The DNA of V-79 Chinese hamster cells was examined by alkaline elution following treatment of cultures with eight different nitrosoureas. Drug incubations were performed under consistent biological conditions of equal toxicity and equal mutation induction at the hypoxanthineguanine phosphoribosyltransferase locus. The goals of this study were to determine whether DNA damage could be detected in cells treated with biologically relevant doses of nitrosoureas and to determine whether the type and number of observed DNA lesions could be correlated with the cytotoxic and mutagenic effects of the drugs. All of the compounds tested produced, to some degree, lesions that were observed as DNA strand breaks upon exposure of the DNA to alkali. The levels of DNA strand breaks and/or alkali-labile lesions were comparable for all of the drugs at the equimutagenic doses. DNA cross-linking was observed at both the equitoxic and the equimutagenic concentrations of the haloethylnitrosoureas, but cross-linking was not observed with methylnitrosourea or streptozotocin. Methylnitrosourea and streptozotocin required approximately 40 times the drug concentration to produce toxicity equal to the haloethylnitrosoureas. These data suggest that the ability to cross-link DNA confers increased cytotoxicity to the haloethylnitrosoureas.

Differential inhibition of the rejoining of X-ray-induced DNA strand breaks in normal and transformed human fibroblasts treated with 1,3-bis(2-chloroethyl)-1-nitrosourea in vitro.

The effects of 1,3-bis(2-chloroethyl)-1-nitrosourea on the rejoining of X-ray-induced DNA strand breaks were examined in normal human fibroblasts (WI-38) and a simian virus 40-transformed derivative (VA-13) with the use of alkaline sucrose sedimentation. 1,3-Bis(2-chloroethyl)-1-nitrosourea was capable of partially inhibiting repair of X-ray-produced DNA strand breaks in both cell types when the drug was added to the culture medium immediately after X-irradiation. However, when 1,3-bis(2-chloroethyl)-1-nitrosourea exposure preceded X-ray by 1 hr, DNA repair was inhibited to a much greater extent than it was when 1,3-bis(2-chloroethyl)-1-nitrosourea followed X-ray. The inhibition of DNA repair by 1,3-bis(2-chloroethyl)-1-nitrosourea appeared to be complete in the transformed VA-13 cells, while only partial inhibition of repair was observed in the normal WI-38 cells.