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Background: Few studies have assessed the prevalence and mortality of simple or complex congenital heart diseases (CHD) in newborns. In Latin America and Caribbean (LAC), CHD epidemiology seems highly variable, with few population-based assessments and different methodologies between studies. To date, the situation in French Guiana, a French overseas territory located in South America between Brazil and Suriname, has never been described., Methods: We analysed CHD prevalence, characteristics and related infant mortality in French Guiana, with a population-based registry analysis of all fetal and live birth CHD cases in infants under 1 year (January 2012-December 2016)., Findings: Overall, 33,796 births (32,975 live births) were registered, with 231 CHD (56 fetuses), including 215 live births. Most frequent CHD categories were anomalies of the ventricular outflow tract and extra-pericardial trunks, and ventricular septal defects. 18.6% (43/231) chromosomal or genetic anomalies, and 6.5% (15/231) terminations of pregnancy were observed. Total CHD prevalence was 68.4 [95% CI: 67.9-68.8] per 10,000, while live birth prevalence was 65.2 [95% CI: 64.7-65.7] per 10,000. Total infant mortality was 9.4/10,000 live births [95% CI 9.1-9.7], with highest rates for functionally univentricular hearts (FUH)., Interpretation: A distinct profile for CHD is highlighted in French Guiana with elevated mortality linked to FUH. A potential determinant of the recognized excess mortality risk might be the presence of chromosomal or genetic anomalies in about a fifth of all CHD. This helps us to better understand CHD burden in this part of South America and provides future keys towards reducing CHD-related infant mortality., Funding: The authors received no financial support for the present research, authorship, and/or publication of this article., Competing Interests: The authors declare no conflicts of interest with respect to the present research, authorship and/or publication of this article., (© 2023 The Author(s).)

Introduction: Several studies have reported a higher frequency and greater morbidity and mortality of multisystem inflammatory syndrome in children (MIS-C) of black African descent., Objectives: We aimed to describe the clinical, laboratory and echocardiographic characteristics as well as outcomes of children with MIS-C requiring admission to a pediatric intensive care unit (PICU) in the French West Indies (FWI), where the majority of the population is Afro-Caribbean., Methods: Ambidirectional observational cohort study between April 1, 2020 and August 31, 2022. Children (age ≤18 years) with MIS-C and organ failure were included. Every patient was monitored and treated following the same protocol, with repeated biological tests, echocardiography, intravenous steroids and polyvalent immunoglobulins. The primary outcomes were clinical, laboratory and echocardiography characteristics., Results: Forty children (median age 7 years, range: 5-11) were included. The majority (77 %) were included prospectively. Thirty-five (87 %) had gastrointestinal symptoms, 30 (75 %) presented initial heart failure (with persisting diastolic dysfunction at day 7) and 18 (45 %) had pericarditis. Sixteen (40 %) were in cardiogenic shock and required inotropic support. Median duration of inotropic support and hospitalization in PICU were respectively 4 and 5 days. The evolution curves of the inflammatory variables matched after treatment. The clinical outcomes were favorable. The Delta variant was associated with the highest incidence of MIS-C., Conclusion: This is the first description of MIS-C course among children of Afro-Caribbean descent. The outcomes were good, without any death or cardiac sequelae. Our work does not support an ethnic susceptibility for severity of MIS-C in Afro-Caribbean population., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Importance: Surgical correction of congenital heart defects (CHDs) has improved the lifespan and quality of life of pediatric patients. The number of congenital heart surgeries (CHSs) in children has grown continuously since the 1960s. This growth has been accompanied by a rise in the incidence of postoperative heart block requiring permanent pacemaker (PPM) implantation., Objective: To assess the trends in permanent pacing after CHS and estimate the economic burden to patients and their families after PPM implantation., Design, Setting, and Participants: In this economic evaluation study, procedure- and diagnosis-specific codes within a single-institution database were used to identify patients with postoperative heart block after CHS between January 1, 1960, and December 31, 2018. Patients younger than 4 years with postoperative PPM implantation were selected, and up to 20-year follow-up data were used for cost analysis based on mean hospital event charges and length of stay (LOS) data. Data were analyzed from January 1, 2020, to November 30, 2022., Exposure: Implantation of PPM after CHS in pediatric patients., Main Outcomes and Measures: Annual trends in CHS and postoperative PPM implantations were assessed. Direct and indirect costs associated with managing conduction health for the 20 years after PPM implantation were estimated using Markov model simulation and patient follow-up data., Results: Of the 28 225 patients who underwent CHS, 968 (437 female [45.1%] and 531 male [54.9%]; 468 patients aged <4 years) received a PPM due to postoperative heart block. The rate of CHS and postoperative PPM implantations increased by 2.2% and 7.2% per year between 1960 and 2018, respectively. In pediatric patients younger than 4 years with PPM implantation, the mean (SD) 20-year estimated direct and indirect costs from Markov model simulations were $180 664 ($32 662) and $15 939 ($1916), respectively. Using follow-up data of selected patients with clinical courses involving 1 or more complication events, the mean (SD) direct and indirect costs were $472 774 ($212 095) and $36 429 ($16 706), respectively., Conclusions and Relevance: In this economic evaluation study, the cost of PPM implantation in pediatric patients was found to accumulate over the lifespan. This cost may represent not only a substantial financial burden but also a health care burden to patient families. Reducing the incidence of PPM implantation should be a focused goal of CHS.

Purpose: Damage to the cardiac conduction system is a major risk of congenital cardiac surgery. Localization of the conduction system is commonly based on anatomic landmarks, which are variable in congenital heart diseases. We introduce a novel technique for identification of conduction tissue regions based on real-time fiberoptic confocal microscopy., Description: We developed a fiberoptic confocal microscopy-based technique to document conduction tissue regions and deployed it in pediatric patients undergoing repair of common congenital heart defects. The technique applies clockface schematics for intraoperative documentation of the location of conduction tissue regions., Evaluation: We created clockface schematics for 11 patients with ventricular septal defects, 6 with tetralogy of Fallot, and 10 with atrioventricular canal defects. The approach revealed substantial variability in the location of the conduction system in hearts with congenital defects. The clockface schematics were used to create plans for subsequent surgical repair., Conclusions: The clockface schematic provides a reliable fiducial system to document and communicate variability of conduction tissue regions in the heart and applies this information for decision-making during congenital cardiac surgery., (Copyright © 2022 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Radiation therapy for the treatment of thoracic malignancies has improved significantly by directing of the proton beam in higher doses on the targeted tumor while normal tissues around the tumor receive much lower doses. Nevertheless, exposure of normal tissues to protons is known to pose a substantial risk in long-term survivors, as confirmed by our work in space-relevant exposures of murine lungs to proton radiation. Thus, radioprotective strategies are being sought. We established that LGM2605 is a potent protector from radiation-induced lung toxicity and aimed in the current study to extend the initial findings of space-relevant, proton radiation-associated late lung damage in mice by looking at acute changes in human lung. We used an ex vivo model of organ culture where tissue slices of donor living human lung were kept in culture and exposed to proton radiation. We exposed donor human lung precision-cut lung sections (huPCLS), pretreated with LGM2605, to 4 Gy proton radiation and evaluated them 30 min and 24 h later for gene expression changes relevant to inflammation, oxidative stress, and cell cycle arrest, and determined radiation-induced senescence, inflammation, and oxidative tissue damage. We identified an LGM2605-mediated reduction of proton radiation-induced cellular senescence and associated cell cycle changes, an associated proinflammatory phenotype, and associated oxidative tissue damage. This is a first report on the effects of proton radiation and of the radioprotective properties of LGM2605 on human lung., Competing Interests: Melpo Christofidou-Solomidou reports grants from the NIH during the conduct of the study. In addition, Melpo Christofidou-Solomidou has patents No. PCT/US14/41636 and No. PCT/US15/22501 pending and has a founders equity position in LignaMed, LLC. All other coauthors declare no conflict of interest.

Soil moisture influences fluxes of heat and moisture originating at the land surface, thus altering atmospheric humidity and temperature profiles. However, empirical and modeling studies disagree on how this affects the propensity for precipitation, mainly owing to the difficulty in establishing causality. We use Granger causality to estimate the relationship between soil moisture and occurrence of subsequent precipitation over the contiguous United States using remotely sensed soil moisture and gauge-based precipitation observations. After removing potential confounding effects of daily persistence, and seasonal and interannual variability in precipitation, we find that soil moisture anomalies significantly influence rainfall probabilities over 38% of the area with a median factor of 13%. The feedback is generally positive in the west and negative in the east, suggesting dependence on regional aridity., (Copyright © 2016, American Association for the Advancement of Science.)

Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway., (©2015 American Association for Cancer Research.)

Radiation therapy (RT) plays a critical role in the local-regional control of head and neck squamous cell carcinoma (HNSCC). However, the efficacy of RT in treating HNSCC is limited by severe normal tissue toxicity, predominantly mucositis. One pharmacological approach for increasing the clinical response to RT is the use of radiation response modifiers that preferentially sensitize tumor cells. Previously we demonstrated that curcumin, a natural plant polyphenol, increased the radiation sensitivity of HNSCC cells and that the observed sensitization was dependent on curcumin-mediated inhibition of thioredoxin reductase 1 (TxnRd1) a key cytosolic regulator of redox-dependent signaling. Here, we examined curcumin-induced radiation sensitization in HNSCC cell lines with differing HPV status and expressing different levels of TxnRd1, in vitro. The intrinsic radiation resistance of the HPV (-) cell lines was significantly higher than the HPV (+) cell lines used in our study. Notably, all of the HPV (-) cell lines expressed high levels of TxnRd1 and exhibited higher intrinsic resistance to RT. While curcumin was effective at increasing the radiation response of the resistant HPV (-) cell lines it had no effect on the HPV (+) cells. Based on these findings we employed an orthotopic, HPV (-) HNSCC tumor model in athymic nude mice to examine the effect of combining curcumin with fractionated RT, in vivo. The combination of curcumin feeding and fractionated RT had a significant effect on tumor doubling time and overall animal survival. We therefore propose that curcumin and RT should be considered as a first line treatment of HPV (-) HNSCC.

The habenula and interpeduncular nucleus (IPN) are part of a dorsal diencephalic conduction system which receives input from cholinergic, striatal, and hypothalamic areas, and sends output to several, disparate midbrain regions. These output regions include the dorsal tegmental nucleus, which is part of a navigation-related system that provides a signal for directional heading. The habenula and IPN also project to the dorsal and medial Raphe nuclei, thought to be involved in mood and behavioral state regulation. Here, cells in both the habenula and IPN were recorded in freely moving rats while they foraged for food pellets. There were four major findings. First, many of the cells tended to fire in sporadic bouts of relatively high versus low rates, and this may be related to intrinsic cell properties discovered during in vitro studies. Second, although these regions are connected to the direction signaling circuit, they do not, themselves demonstrate a directional signal. Third, about 10% of the cells in the lateral habenula showed a strong correlation between rate and angular head motion. This may constitute an important, requisite input to the above-mentioned head direction circuit. Finally, many of the cells in each region showed a temporally coarse correlation with running speed, so that bouts of high frequency firing coincided with episodes of higher behavioral activation. This last finding may be related to work which shows an influence of the habenula on locomotor activity, and in relation to the protective effects of exercise in relation to stress, as mediated by the Raphe nuclei.

The promyelocytic leukemia (PML) gene, a tumor suppressor inactivated in acute promyelocytic leukemia (APL), regulates apoptosis induced by DNA damage. However, the molecular mechanisms by which PML modulates apoptosis following genotoxic stress are only partially elucidated. PML is essential for p53-dependent induction of programmed cell death upon gamma-irradiation through PML-nuclear body (NB)-mediated control of p53 acetylation. Here, we show that PML selectively regulates proapoptotic transcription factors upon different types of DNA damage. We find that Pml inactivation protects fibroblasts from UV-induced apoptosis in a p53-independent manner. We demonstrate that c-Jun is required for UV-induced apoptosis and that PML is essential for both c-Jun transcriptional activation and DNA binding upon UV radiation. We find that PML physically interacts with c-Jun and that upon UV radiation the PML-NBs reorganize into novel nuclear microspeckled structures (UV-NBs), where PML and c-Jun dynamically accumulate. These data identify a novel PML-dependent pathway for c-Jun transcriptional activation and induction of apoptosis in response to DNA damage and shed new light on the role of PML in tumor suppression.

We demonstrate, theoretically, that oxygen diffusion distance is related to the metabolic rate of tumors (QO2) as well as the oxygen tension. The difference in QO2 rate between tumors can vary by as much as 80-fold. Inhibition of oxygen utilization by glucose or chemical inhibitors can improve the diffusion distance. Combining respiratory inhibitors with increased availability of oxygen will further improve the oxygen diffusion distance for all tumors. A simple means for inhibiting oxygen consumption is the use of glucose (the Crabtree effect). The inhibition of tumor oxygen utilization by glucose occurs in R323OAc mammary carcinoma and 9L glioma cells. However, stimulation of oxygen consumption is observed with glucose in the Q7 hepatoma cell line. MIBG, a known inhibitor of oxygen utilization, blocks oxygen consumption in 9L, but is weakly inhibitory with the Q7. Q7 tumor cells demonstrate an anomalous behavior of glucose and MIBG on oxygen consumption. Our results clearly demonstrate the necessity for comparing effects of different agents on different tumor cells. Generalizations cannot be made with respect to the choice of inhibitor for in vivo use. Our work shows that oxygen consumption also can be inhibited with malonate and chlorosuccinate. These substrates may be effective in vivo, where glucose is low and glutamine is the major substrate. Our results indicate that information about individual tumor substrate-linked metabolic controls may be necessary before attempting to inhibit oxygen utilization in vivo for therapeutic benefit.

Glutathione depletion by L-buthionine sulfoximine inhibits the growth of Ehrlich mouse mammary carcinoma, R3230Ac rat mammary carcinoma and the PC3 human prostrate carcinoma cells, in vitro. Inhibition of growth occurs within the first 24 hours after exposure to the drug. The cell density does not increase over the initial cell density over 7 days. A549 human lung carcinoma and the DU145 human prostrate carcinoma cells show no inhibition of growth under the same treatment conditions. A comparative study of the R323OAc and A549 cells demonstrated a marked increase in apoptosis following L-BSO treatment in R3230Ac, which was dependent on L-BSO concentration and incubation time. L-BSO did not induce apoptosis in A549 cells at any of the concentrations tested. The incidence of apoptosis for R323OAc cells following exposure to 0.1 mM L-BSO was similar to the incidence of radiation-induced apoptosis observed after exposure to 10 Gy. Treatment with L-BSO or radiation alone inhibited O2 utilization in of R323Oac, while no effect on O2 utilization was observed in A549 cells. LBSO altered the bioreductive capacity of both the R323OAc and A549 cells. These results suggest that the ability of L-BSO to block mitochondrial O2 utilization may be involved in the apoptotic response in R3230Ac cells.

Recent findings indicate a different role of the left and right hippocampal formation (RHF) in homing pigeon navigational map learning. However, it remains uncertain whether the left or the RHF may play a more important role in navigation based on familiar landmarks. In the present study, we attempted to answer this question by experimentally releasing control and left and right hippocampal ablated pigeons from familiar training sites under anosmia, to render their navigational map dysfunctional, and after a phase-shift of the light-dark cycle, to place into conflict a pilotage-like landmark navigational strategy and a site-specific compass orientation landmark navigational strategy. Both left and right hippocampal ablated birds succeeded in learning to navigate by familiar landmarks, and both preferentially relied on sun-compass based, site-specific compass orientation to home. Like bilateral hippocampal lesioned birds, and in contrast to intact controls, neither ablation group adopted a pilotage-like strategy. We conclude that both the left and RHF are necessary if pilotage-like, familiar landmark navigation is to be learned or preferentially used for navigation.

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related., (Copyright 2000 Academic Press.)

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.

Purpose: At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells., Results: Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC's ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells., Conclusions: Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

The kinetics of the production of hydroxyl radicals during the autoxidation of ferrous ion complexes at pH 7.4 was investigated using the fluorescent probe coumarin-3-carboxylic acid. Polyphosphates (tri- and tetrapolyphosphate and their adenosine derivatives), citrate, and acetic derivatives of ethyleneamine ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), ethylenediamine-(N,N')-diacetic acid (EDDA) and nitrilotriacetic acid (NTA) were used as iron chelators. Production of hydroxyl radical in these chemical systems was compared with that by radiation to determine the equivalent doses of radiation that produced equal amounts of .OH. The amount of .OH formed during ferrous ion autoxidation is determined by the concentration of the complex, its structure and the radical scavenging by the chelator molecule. Production of .OH for homologous ethylenamine acetates increases with increased complex stability: NTA < EDDA < TTHA < EDTA < DTPA. The radiation dose equivalence for 0.1 mM complexes increased from 5 Gy for NTA to 25 Gy for DTPA. The radiation dose equivalence for polyphosphates was 15 Gy for tripolyphosphate and 32 Gy for tetrapolyphosphate. The dose equivalences for adenosine phosphates are lower, 5 Gy for ATP and 10 Gy for adenosine tetraphosphate, due to intramolecular .OH scavenging. The rate of generation of .OH shows an inverse correlation with the charge of the ferrous ion complex, varying from 2 cGy/s for DTPA to 1.2 Gy/s for EDTA. The data presented indicate the usefulness of autoxidation of ferrous ion complexes for generation of .OH in chemical systems. The ability to control the amount and the rate of production of .OH may prove useful for examining the cytotoxic effects of .OH generated in biological systems.

We found an unique effect of oncogene transfections on rat embryo cell (REF) respiration, glycolysis and radiation response. Radioresistance, defined as an increase in Do, increases for REF cells transfected with v-myc or H-ras oncogenes. The combination of both oncogenes confers the maximal radioresistance. Our work shows inhibition of oxygen uptake when cells are transfected with v-myc or H-ras alone. However, oxygen uptake increases when cells are transfected simultaneously with v-myc + H-ras (3.7,2.1,2.8). A higher oxygen consumption results from increased utilization of pyruvate via the Kreb's cycle. Succinate stimulates cellular oxygen consumption. The maximum stimulation of oxygen consumption by succinate occurred with v-myc + H-ras transfected cells. The glycolysis of the transfected cells is also altered by the oncogenes. Our glycolytic measurements indicate the H-ras oncogene causes the largest stimulation of glycolysis. Our data shows that transfection with oncogenes has a major effect on cellular glycolysis, oxidative metabolism as well as the subsequent radiation response.

Hydroperoxides are reduced in mammalian cells by a coupled enzyme pathway involving glutathione peroxidase, glutathione reductase and the oxidative limb of the pentose cycle. Oxidation of glucose-6-phosphate by the pentose cycle yields two molecules of NADPH, which can reduce two hydroperoxide molecules to the corresponding alcohol. Rat embryo fibroblasts (REF) transfected with v-myc reduce hydroperoxides slower than the primary REF cell line-measured both as real time peroxide loss and as increased glucose oxidation via the pentose cycle. The v-myc transfected cell line is 50-fold more sensitive to the toxic effects of tBu-OOH. The decreased reduction of peroxides by v-myc transfected cells is not due to changes in the activities of GSH reductase or the enzymes of the oxidative pentose cycle, since diamide stimulates PC activity equally in both cell lines. In addition, the activities of these enzymes, measured in cell homogenates do not differ significantly between the cell lines. Also total GSH peroxidase activity, assayed in cell homogenates, is not significantly different between the cell lines. Two human tumour cell lines which overexpress myc family proteins: NCI-H69, a small-cell lung cancer line which expresses elevated levels of N-myc, and HL-60 cells which overexpress c-myc, also exhibit low levels of pentose cycle stimulation in the presence of tBu-OOH, and a decreased capacity to reduce hydrogen peroxide by peroxide electrode.

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Purpose: Metal chelating agents and antioxidants were evaluated as potential protectors against aerobic SR 4233 cytotoxicity in Chinese hamster V79 cells. The differential protection of aerobic and hypoxic cells by two metal chelators, desferrioxamine and Tiron, is discussed in the context of their potential use in the on-going clinical trials with SR 4233., Methods and Materials: Cytotoxicity was evaluated using clonogenic assay. SR 4233 exposure was done in glass flasks as a function of time either alone or in the presence of the following agents: superoxide dismutase, catalase, 5,5-dimethyl-1-pyrroline, Trolox, ICRF-187, desferrioxamine, Tiron (1,2-dihydroxybenzene-3,5-disulfonate), and ascorbic acid. Experiments done under hypoxic conditions were carried out in specially designed glass flasks that were gassed with humidified nitrogen/carbon dioxide mixture and with a side-arm reservoir from which SR 4233 was added to cell media after hypoxia was obtained. Electron paramagnetic resonance studies were also performed., Results: Electron paramagnetic resonance and spectrophotometry experiments suggest that under aerobic conditions SR 4233 undergoes futile redox cycling to produce superoxide. Treatment of cells during aerobic exposure to SR 4233 with the enzymes superoxide dismutase and catalase, the spin trapping agent DMPO, the water-soluble vitamin E analog Trolox, and the metal chelator ICRF-187 provided little or no protection against aerobic SR 4233 cytotoxicity. However, two other metal chelators, desferrioxamine and Tiron, afforded significant protection against aerobic SR 4233 cytotoxicity (protection factors at 50% survival were 3.8 and 3.1, respectively), while exhibiting minimal protection to hypoxic cells treated with SR 4233., Conclusions: One potential mechanism of aerobic cytotoxicity is redox cycling of SR 4233 with molecular oxygen resulting in several potentially toxic oxidative species that overburden the intrinsic intracellular detoxification systems such as superoxide dismutase, catalase, and glutathione peroxidase. This study identifies two metal chelating agents, desferrioxamine and Tiron, that were able to protect against aerobic but not hypoxic SR 4233 cytotoxicity.

Purpose: Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity., Methods and Materials: SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In vivo the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled 14CO2 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay., Results: SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H2O2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity., Conclusion: Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.

We have shown previously that the thiol-containing radioprotector dithiothreitol (DTT) kills V79 cells in a manner that is dependent on both the concentration of DTT and the medium. The results are consistent with the hypothesis that DTT toxicity is caused by the copper-catalyzed oxidation of DTT, forming H2O2, which in turn produces .OH, the ultimate toxic species, via the metal-catalyzed Fenton reaction. Because it is known that the pentose cycle plays a role in the ability of cells to deal with oxidative stress, the hypothesis that the pentose cycle is involved in the response of cells to DTT is tested in this paper. The results show that toxicity of both DTT and H2O2 in V79 cells is greater in cells exposed to the drugs in medium lacking glucose than in cells in medium containing glucose. Addition of glucose to medium or buffer lacking it decreases DTT- and H2O2-induced cell killing. Studies using cells deficient in glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose cycle, show that cells of the mutant cell lines (E16 and E48) are more sensitive to cell killing by both DTT and H2O2 than are the parental CHO K1D cells when exposed to the drugs in medium containing glucose. However, toxicity does not differ significantly among the three cell lines when they are exposed to DTT or H2O2 in phosphate-buffered saline that lacks glucose. Measurements of pentose cycle activity show that the pentose cycle in K1D cells is stimulated by DTT, with the pattern and drug concentration dependence of the stimulation being similar to that for cell killing. However, the pentose cycle is not stimulated by DTT in G6PD-deficient cell lines. The data are consistent with the hypothesis that the pentose cycle is one of the cellular pathways that mediates the oxidative stress imposed by DTT or H2O2.

In this paper we examine the susceptibility of a series of G6PD- CHO cell lines to a variety of chemical oxidants. Addition of these drugs to K1D, the parental cell line, results in as much as a 20-fold increase in pentose cycle (PC) activity over control values. In two of our mutant lines, E16 and E48, little or no stimulation of PC activity is seen. These lines are shown to be much more susceptible to the toxic effects of the chemical oxidants t-butyl hydroperoxide and diamide. PC activity is also stimulated by ionizing radiation in K1D cells. One of the G6PD- cell lines has an increased aerobic radiation response compared to the parental line. However, since this is not the case with the other G6PD- cell lines, it is unclear whether this represents a difference in the absolute value of PC activity or some additional variable that may be influencing the results.

The Bowman Birk protease inhibitor (BBI) has been shown to be an effective suppressor of carcinogenesis in vivo and in vitro. In this report we demonstrate that normal human fibroblasts and Bloom cells contain a BBI-inhibitable proteolytic activity. The enzyme cleaves gelatin, has a molecular mass of 43 kDa, and is located in the cytosol. This activity has maximal activity at pH 8 and was inhibited by diisopropylfluorophosphate but was not affected by EDTA or 1,10-phenanthroline, indicating that this enzyme is a serine protease. We have reported previously that a similar BBI-inhibitable activity is present in C3H/10T1/2 mouse embryo fibroblast cells. Our results suggest that a common "target enzyme" of the BBI is present in mouse and human cells.

A panel of six monoclonal antibodies (MAbs) was employed to evaluate antigen expression in pulmonary adenocarcinomas and mesotheliomas. Monoclonal anti-human milk fat globulin (HMFG-2), anti-carcinoembryonic antigen (NP-2), anti-epithelial membrane antigen (EMA), anti-cytokeratin (PKK-1), anti-tumor-associated antigen 72 (B72.3), and anti-human myelomonocytic antigen (Leu M-1) antibodies were used to localize their respective antigens in formalin-fixed, paraffin-embedded tumors by using the avidin-biotin-complex immunoperoxidase technique. In all, 28 mesotheliomas obtained from Ohio State University Anatomic Pathology files and from a Southwest Oncology Group (SWOG) protocol were compared to 22 pulmonary adenocarcinomas by using this MAb panel. None of the mesotheliomas demonstrated positive staining with MAbs NP-2 (anti-CEA) or Leu M-1. However, 95% (21/22) of adenocarcinomas stained with one of these two antibodies. Although neither of these two MAbs stained all adenocarcinomas, each antibody demonstrated positive immunostaining in more than 90% of the adenocarcinomas studied. Therefore, MABs NP-2 and Leu M-1 are, individually, quite useful for distinguishing mesothelioma from adenocarcinoma. However, in our study, no single MAb could be used to distinguish these two tumor types in every case. MAb B72.3 stained 91% (20/21) adenocarcinomas but also stained 7% (2/28) of mesotheliomas. MAb HMFG-2 reacted positively with 95% of adenocarcinomas, but also stained 39% of the mesotheliomas, usually in a membranous pattern. MAbs EMA and PKK-1 were not found useful in distinguishing mesothelioma from adenocarcinoma. We conclude that MAbs Leu M-1 and NP-2 were both useful in distinguishing mesothelioma from pulmonary adenocarcinoma in that positive staining was demonstrated in adenocarcinomas and not mesotheliomas.

The sensitivity of tumor cells to the killing effects of ionizing radiation is thought to be one of the major determinants of curability of tumors in patients treated with radiation therapy. This paper reviews the evidence from our laboratory and other groups which supports a role for oncogenes in the induction of radioresistance in cultured mammalian cells. Primary rat embryo cells (REC) were chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offered several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of synergism between oncogenes on the induction of the radioresistant phenotype. The results show that the activated H-ras oncogene induces radiation resistance in primary rat cells after transformation, but that the effect of the oncogene itself is small. However, the myc oncogene, which has no effect on radiation resistance by itself, appears to have a synergistic effect on the induction of radiation resistance by H-ras. Radiation resistance induced by H-ras plus myc is characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. The AdenoE1A oncogene which will also act synergistically with ras in transformation assays plays a less clear-cut role in assays of radiation resistance. The H-ras oncogene is also known not only to transform cells but also to induce metastatic behavior in the tumors which form after these transformed cells are injected into syngeneic animals or nude mice. We have also shown in our primary rat embryo cell system that the induction of metastatic behavior in transformed cells, like the induction of radioresistance depends on a complex interaction between oncogenes and the cellular background. This evidence will be reviewed to demonstrate some of the analogies between radiation resistance and metastasis as examples of the complex alterations in cellular phenotype which occur after oncogene transfection.(ABSTRACT TRUNCATED AT 400 WORDS)

A new intraoperative approach to tumor localization using radiolabelled monoclonal antibody (MAb) B72.3 involves the use of a hand-held gamma-detecting probe (GDP) by the surgeon and, subsequently, the pathologist. We report here the use of 125I-labelled MAb B72.3 IgG and a GDP to localize primary and metastatic colorectal cancer in 31 patients. The patients were administered radiolabelled MAb i.v., and all underwent surgical exploration 5 to 35 days post-injection. In vivo localization of the MAb was evaluated using a GDP, with tumor and normal tissue counts being obtained. In each case, the subsequent tumor and normal tissue that were resected were analyzed in vitro for MAb localization; this was evaluated by calculating the radiolocalization index, i.e., the ratio of the injected dose per gram localized to tumor versus that of normal tissue. When the GDP was used intraoperatively, MAb B72.3 localized tumors in 68% (21/31) of the patients; the arbitrary criterion of tumor-to-normal tissue ratios higher than or equal to 2.0:1 in vivo being taken as positive. Resected tumor radiolocalization indices ranged from 0.5 to 543, and 71% (22/31) of the patients studied had tumors with radiolocalization indices higher than or equal to 3. Of 50 carcinoma biopsies, 34 that were probe-positive were antigen-positive when B72.3 was used in immunoperoxidase assays, while 4 carcinoma biopsies that were probe-negative were also antigen-negative. Twelve of 50 biopsies were probe-negative and antigen-positive, but many of these lesions only contained a few antigen-positive cells; none of the 50 was probe-positive and antigen-negative. Tumors of all histologic grades localized injected MAb and, in general, higher in vivo probe ratios and radiolocalization indices were obtained from patients who underwent surgery 20 to 35 days following injection of the MAb. MAb B72.3 localized tumor in all sites to which colon carcinoma commonly metastasizes, including mesenteric and peri-aortic lymph nodes, liver, lung, and peri-rectal soft tissue. There was a strong statistical correlation (p = 0.001) between detecting MAb B72.3 localization to tumors using the GDP intraoperatively and subsequent in vitro analysis of cpm/g for tumor versus normal tissues. These studies thus further validate the use of 125I-labelled MAb B72.3 IgG and of a hand-held gamma probe for the intraoperative detection of carcinomatous lesions.

Misonidazole, SR-2508, nitrofurazone and other nitroheterocycles stimulated release of 14CO2 from [1-14C]glucose but not from [6-14C]glucose when incubated with mouse Ehrlich ascites cells or human A549 lung carcinoma cells in vitro. This demonstrated that the nitro compounds activated the hexose monophosphate shunt and is evidence that an important pathway of nitro reduction in these cell lines is electron transfer from NADPH-dependent cytochrome c reductase to the nitro group. Shunt activity was stimulated under both aerobic and anaerobic conditions. For catalase-free Ehrlich cells, aerobic effects were greater than anaerobic, indicating that NADPH was used for reduction of H2O2, via GSH peroxidase and reductase, as well as for one-electron nitro reduction, under aerobic conditions. Several of the compounds tested stimulated 14CO2 release from [2-14C]glucose as well as from [1-14C]-glucose. This shows that the cellular requirement for NADPH, in the presence of nitro drug, was great enough to cause recycling of pentose phosphates. Recycling could decrease the availability of ribose-5-P needed for nucleic acid synthesis, which could partly explain the inhibition of DNA synthesis observed upon prolonged aerobic incubation of cells with nitro compounds. Comparison of the rate of disappearance of nitrofurazone from anaerobic A549 cell suspensions with the rate of 14CO2 release suggests that the drug reduction in this cell line was catalyzed almost entirely by NADPH-requiring enzymes.

Alveolar soft part sarcoma is a soft tissue neoplasm of unknown histogenesis which has a distinctive morphology. It is a relatively rare tumor with approximately 200 cases described in the literature. Only two cases have previously been reported as occurring in the vagina. The purpose of this paper is to report the third case of alveolar soft part sarcoma occurring in the vagina and to emphasize the role of electron microscopy in the differential diagnosis.