Temporal lobe epilepsy and affective disorders: the role of the subgenual anterior cingulate cortex.

OBJECTIVE: Reduced deactivation within the default mode network (DMN) is common in individuals with primary affective disorders relative to healthy volunteers (HVs). It is unknown whether similar network abnormalities are present in temporal lobe epilepsy (TLE) patients with a history of affective psychopathology. METHODS: 17 TLE patients with a lifetime affective diagnosis, 31 TLE patients with no formal psychiatric history and 30 HVs were included. We used a visuo-spatial 'n-back' paradigm to compare working memory (WM) network activation between these groups. Post hoc analyses included voxel-based morphometry and diffusion tensor imaging. The Beck Depression Inventory-Fast Screen and Beck Anxiety Inventory were completed on the day of scanning. FINDINGS: Each group activated the fronto-parietal WM networks and deactivated the typical DMN in response to increasing task demands. Group comparison revealed that TLE patients with lifetime affective morbidity showed significantly greater deactivation in subgenual anterior cingulate cortex (sACC) than either the TLE-only or the HVs (p<0.001). This effect persisted after covarying for current psychotropic medication and severity of current depressive/anxiety symptoms (all p<0.001). Correlational analysis revealed that this finding was not driven by differences in task performance. There were no significant differences in grey matter volume or structural connectivity between the TLE groups. CONCLUSIONS: Our results provide novel evidence suggesting that affective psychopathology in TLE has a neurobiological correlate, and in this context the sACC performs differently compared with network activity in primary affective disorders.

Neural correlates of working memory in Temporal Lobe Epilepsy--an fMRI study.

It has traditionally been held that the hippocampus is not part of the neural substrate of working memory (WM), and that WM is preserved in Temporal Lobe Epilepsy (TLE). Recent imaging and neuropsychological data suggest this view may need revision. The aim of this study was to investigate the neural correlates of WM in TLE using functional MRI (fMRI). We used a visuo-spatial 'n-back' paradigm to compare WM network activity in 38 unilateral hippocampal sclerosis (HS) patients (19 left) and 15 healthy controls. WM performance was impaired in both left and right HS groups compared to controls. The TLE groups showed reduced right superior parietal lobe activity during single- and multiple-item WM. No significant hippocampal activation was found during the active task in any group, but the hippocampi progressively deactivated as the task demand increased. This effect was bilateral for controls, whereas the TLE patients showed progressive unilateral deactivation only contralateral to the side of the hippocampal sclerosis and seizure focus. Progressive deactivation of the posterior medial temporal lobe was associated with better performance in all groups. Our results suggest that WM is impaired in unilateral HS and the underlying neural correlates of WM are disrupted. Our findings suggest that hippocampal activity is progressively suppressed as the WM load increases, with maintenance of good performance. Implications for understanding the role of the hippocampus in WM are discussed.

Effective medical education: insights from the Cochrane Library.

UNLABELLED: In 2006, the Accreditation Council for Continuing Medical Education highlighted the need for linking educational activities to changes in competence, performance, or patient outcomes. Hence, educational providers increasingly need to know what strategies are effective. The Cochrane Library is widely regarded as the best source of credible evidence concerning health care. The authors searched the Cochrane Database of Systematic Reviews (issue 4 for 2006) using the search terms "continuing medical education," "medical education," and "continuing education." They conducted a second complementary search of this database by review group (Effective Practice and Organization of Care). Finally, the authors examined the references of recent review articles for Cochrane reviews and found 9 relevant reviews. The most effective educational methods were the most interactive. Combined didactic presentations and workshops were more effective than traditional didactic presentations alone. Medical education was more effective when more than 1 intervention occurred, especially if these interventions occurred over an extended period. Targeted education should focus on changing a behavior that is simple, because effect size is inversely proportional to the complexity of the behavior. In the era of evidence-based medicine, interventions-including educational ones-should reflect the best available evidence. Cochrane reviews of randomized controlled trials of educational methods provide important guidance that often challenges traditional didactic approaches. Integrating the findings from the Cochrane reviews may allow continuing medical education to be more successful in bringing about changes to healthcare providers' behavior. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader should be able to explain the scientific evidence concerning the effectiveness of various techniques used for continuing medical education, state the relative value of such techniques as traditional didactic lectures, conferences led by local opinion leaders, interactive workshops, and educational outreach visits, and identify the value and limitations of teaching critical appraisal skills.

Outcome of ethnic minorities with acute or chronic leukemia treated with hematopoietic stem-cell transplantation in the United States.

PURPOSE: We previously reported a higher risk of mortality among Hispanics after allogeneic hematopoietic stem-cell transplantation (HSCT). However, it is not known how specific post-transplantation events (acute or chronic graft-versus-host disease [GVHD], treatment-related mortality [TRM], and relapse) may explain mortality differences. The purpose of this study was to examine the relationship between ethnicity and post-transplantation events and determine their net effect on survival. PATIENTS AND METHODS: We identified 3,028 patients with acute myeloid leukemia, acute lymphoblastic leukemia, or chronic myeloid leukemia reported to the International Bone Marrow Transplant Registry between 1990 and 2000 who received an HLA-identical sibling HSCT after a myeloablative conditioning regimen in the United States. There were 2,418 white patients (80%) and 610 ethnic minority patients (20%), of whom 251 were black (8%), 122 were Asian (4%), and 237 were Hispanic (8%). Cox proportional hazards regression was used to compare outcomes between whites and ethnic minorities while adjusting for other significant clinical factors. RESULTS: No statistically significant differences in the risk of acute or chronic GVHD, TRM, or relapse were found between whites and any ethnic minority group. However, Hispanics had higher risks of treatment failure (death or relapse; relative risk [RR] = 1.30; 95% CI, 1.08 to 1.54; P = .004) and overall mortality (RR = 1.23; 95% CI, 1.03 to 1.47; P = .02). CONCLUSION: The higher risks of treatment failure and mortality among Hispanics may be the net result of modest but not statistically significant increases in both relapse and TRM and cannot be accounted for by any single transplantation-related complication. Further studies should examine the role of social, economic, and cultural factors.

Primary B cell lymphoma of the external auditory canal.

Temporal bone lymphomas are rare and typically metastatic neoplasms. We describe a case of primary B cell lymphoma that originated in the external auditory canal of an elderly woman. The diagnosis was based on histopathologic examination supplemented by immunophenotypic analysis. The patient was treated with external-beam radiation and remained disease-free throughout 9 years of follow-up. We also point out that the presence of non-Hodgkin's lymphoma in an unusual site may be an indication that the patient has an acquired immunodeficiency syndrome.

Factors affecting reorganisation of memory encoding networks in temporal lobe epilepsy.

AIMS: In temporal lobe epilepsy (TLE) due to hippocampal sclerosis reorganisation in the memory encoding network has been consistently described. Distinct areas of reorganisation have been shown to be efficient when associated with successful subsequent memory formation or inefficient when not associated with successful subsequent memory. We investigated the effect of clinical parameters that modulate memory functions: age at onset of epilepsy, epilepsy duration and seizure frequency in a large cohort of patients. METHODS: We studied 53 patients with unilateral TLE and hippocampal sclerosis (29 left). All participants performed a functional magnetic resonance imaging memory encoding paradigm of faces and words. A continuous regression analysis was used to investigate the effects of age at onset of epilepsy, epilepsy duration and seizure frequency on the activation patterns in the memory encoding network. RESULTS: Earlier age at onset of epilepsy was associated with left posterior hippocampus activations that were involved in successful subsequent memory formation in left hippocampal sclerosis patients. No association of age at onset of epilepsy was seen with face encoding in right hippocampal sclerosis patients. In both left hippocampal sclerosis patients during word encoding and right hippocampal sclerosis patients during face encoding, shorter duration of epilepsy and lower seizure frequency were associated with medial temporal lobe activations that were involved in successful memory formation. Longer epilepsy duration and higher seizure frequency were associated with contralateral extra-temporal activations that were not associated with successful memory formation. CONCLUSION: Age at onset of epilepsy influenced verbal memory encoding in patients with TLE due to hippocampal sclerosis in the speech-dominant hemisphere. Shorter duration of epilepsy and lower seizure frequency were associated with less disruption of the efficient memory encoding network whilst longer duration and higher seizure frequency were associated with greater, inefficient, extra-temporal reorganisation.

The reaction of xanthine oxidase with aldehydic products of lipid peroxidation.

A quantitative structure-activity relationship for the reaction of xanthine oxidase with a homologous series of alpha, beta-unsaturated aldehydes, which are known to be products of lipid peroxidation, was investigated. Aldehydes in the series 2-butenal through 2-nonenal and 4-hydroxy-2-nonenal, displayed differential reactivity toward xanthine oxidase as measured by production of the superoxide radical anion. Kinetic parameters for the rate of superoxide production and substrate affinity were determined via the superoxide dismutase-sensitive reduction of cytochrome c. Trends in kinetic parameters as a function of carbon number for the series of trans-2-enals was consistent with a dependence on substrate hydrophobicity. Log kw', a hydrophobicity constant widely employed as a model for the octanol/water partition coefficient, was determined by reversed phase liquid chromatography for the alpha, beta-unsaturated aldehydes in this study. Linear relationships for the correlation of substrate binding (pKm) and efficiency of superoxide production (log kcat/Km) with substrate hydrophobicity (log kw') were found. The mode of inhibition of xanthine oxidation by 2-butenal is shown to be noncompetitive, suggesting distinct binding sites for purine and aldehydic substrates. It is suggested that the reaction of xanthine oxidase with unsaturated aldehydes could be an important route of amplification of oxidative damage in cells.

Beta carotene and its oxidation products have different effects on microsome mediated binding of benzo[a]pyrene to DNA.

The effects of beta-carotene (betaC) and its oxidation products on the binding of benzo[a]pyrene (BaP) metabolites to calf thymus DNA was investigated in the presence of rat liver microsomes. Mixtures of betaC oxidation products (betaCOP) as well as separated, individual betaC oxidation products were studied. One set of experiments, for example, involved the use of the mixture of betaCOP obtained after a 2-h radical-initiated oxidation. For this data set, the incorporation of unoxidized betaC into microsomal membranes caused the level of binding of BaP metabolites to DNA to decrease by 29% over that observed in the absence of betaC; however, the incorporation of the mixture of betaCOP caused the binding of BaP metabolites to DNA to increase 1.7-fold relative to controls without betaC. Two variations of this experiment were studied: (1) When no NADPH was added, betaC decreased the binding of BaP metabolites to DNA by 19%, but the mixture of betaCOP increased binding by 3.3-fold relative to that observed in the absence of betaC. (2) When NADPH was added under near-anaerobic conditions, betaC caused an almost total (94%) decrease in binding whereas betaCOP had no effect on the amount of binding relative to that observed in the absence of betaC. Both betaCOP and cumene hydroperoxide caused BaP metabolites to bind to DNA even when NADPH was omitted from the incubation mixture. Separation of the mixture of betaC oxidation products into fractions by HPLC allowed preliminary testing of individual betaC oxidation products separately; of the various fractions tested, the products tentatively identified as 11,15'-cyclo-12,15-epoxy-11,12,15,15'-tetrahydro-beta-carotene and beta-carotene-5,6-epoxide appeared to cause the largest increase in BaP-DNA binding. Microsomes from rats induced with 3-methylcholanthrene (3MC) or Aroclor 1254 produced different levels of binding in some experimental conditions. We hypothesize that, under some conditions, the incorporation of betaC into microsomal membranes can be protective against P450-catalyzed BaP binding to DNA; however, the incorporation of betaCOP facilitates the formation of BaP metabolites that bind DNA, although only certain P450 isoforms catalyze the binding process.

Single electron reduction of xenobiotic compounds by glucose oxidase from Aspergillus niger.

Various species of fungi express glucose oxidase that catalyzes formation of gluconolactone from glucose with concomitant, direct divalent reduction of molecular oxygen to hydrogen peroxide. A physiological function ascribed to this extracellular enzyme is production of hydrogen peroxide for use in lignin degradation catalyzed by lignin peroxidases. Herein, we show that glucose oxidase can catalyze one-electron reduction of several different classes of xenobiotic compounds resulting in generation of free radical products. Electron spin resonance (ESR) spectroscopy was used to visualize the one-electron reduction products of 4-nitropyridine-N-oxide (4NPO), 1,4-naphthoquinone (1,4NQ), and dichlorophenolindolphenol (DCPIP). Hyperfine splitting constants were used to generate computer simulations of the spectra confirming the presence of free radical products.

tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria.

When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration.

A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids.

Herein, we report a new, rapid,and reliable method for measuring the protective antioxidant potential of pure antioxidant solutions or biological tissues. Peroxyl radicals generated by thermal homolysis of 2,2'-azobis-amidinopropane (ABAP) cause the oxidation of alpha-keto-gamma-methiolbutyric acid (KMBA) to ethylene; ethylene formation is monitored by gas chromatographic analysis of head space from the reaction vessel. The partial inhibition of ethylene formation in the presence of antioxidants that compete with KMBA for oxyradicals is the basis of the Total Oxyradical Scavenging Capacity Assay (TOSCA). The assay is shown to be reliable for quantifying ROS scavenging potential. The quantifiable parameters are consistent with the relative order of those predicted by the fluorescence- and oxygen electrode-based assays reported in the literature. Antioxidants competing for peroxyl radicals influenced the rate of KMBA oxidation in different ways, but the calculation of TOSC was not affected by such variations. Responses were linear over a wide range of sample concentrations and the TOSC values of classical soluble antioxidants showed the following relative order: Trolox > uric acid > ascorbic acid > GSH. The KMBA method was reliable for biological tissues; the TOSC for 1 microg rat liver cytosolic protein was 0.40 +/- 0.02 and for the microsomal membrane, 0.15 +/- 0.03. Soluble antioxidants accounted for 77% of the protective antioxidant potential in rat liver cytosol. When incorporated into the microsomal membrane, alpha-tocopherol markedly enhances antioxidant protection against peroxyl radical; thus, the assay is suitable for the assessment of fat-soluble antioxidants.

Effects of chronic ethanol ingestion on liver glycogen phosphorylase in male and female rats.

The effects of chronic ethanol ingestion on a preparation of liver glycogen phsophorylase have been studied. A coupled assay in the direction of glycogenolysis was used. In the absence of AMP, a significant decrease in specific activity was observed in both males (19%) and females (30%). AMP additions stimulated phosphorylase activity and completely obliterated the ethanol-induced decreases in both sexes of animal. Kinetic studies, done in the absence of AMP, showed that only the apparent Vmax had been altered by ethanol. These data suggest that decreases in liver glycogen after chronic ethanol ingestion may not be related to the specific activity of glycogen phosphorylase. Using both glucose and caffeine as negative effectors, addditional studies demonstrated that the inhibitory effects of caffeine had been altered by ethanol in both males and females and that the inhibitory effects of glucose had been altered only in females. Even though the specific activity for phosphorylase did not directly implicate this enzyme in the ethanol-induced decrease in liver glycogen stores, the latter data regarding glucose and caffeine suggest that chronic ethanol ingestion has altererd this enzyme and that differences exist between males and females.

Differential effects of the cytochrome P-450/reductase ratio on the oxidation of ethanol and the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA).

NADPH-cytochrome P-450 reductase catalyzes a low rate of oxidation of hydroxyl radical scavenging agents such as ethanol and 2-keto-4-thiomethylbutyric acid (KMBA), in a reaction markedly stimulated by the addition of ferric-EDTA. The effect of various ratios of cytochrome P-450 (phenobarbital-inducible isozyme)/reductase on the oxidation of ethanol and KMBA was determined: There was essentially no increase in KMBA oxidation over the range of ratios from 0.5 to 5 as compared to the reductase-catalyzed rate. High ratios actually caused some decrease in KMBA oxidation, which was especially notable under conditions of increased rates of hydroxyl radical generation (presence of increasing amounts of ferric-EDTA). This decrease at high P-450/reductase ratios could reflect tight coupling of reductase to P-450-PB, therefore decreasing electron transfer from reductase to ferric-EDTA, or could involve non-specific scavenging of .OH by P-450-PB. Indeed, native, but not boiled, P-450 inhibited KMBA oxidation by the xanthine oxidase system. By contrast, the oxidation of ethanol was stimulated at all concentrations of P-450-PB, and this increase was not sensitive to desferrioxamine. However, under conditions of high rates of .OH production, the ethanol oxidation profile tended to resemble the KMBA oxidation profile with respect to the effect of P-450-PB, whereas the two profiles were different under conditions of low rates of .OH production. These results suggest that P-450-PB does not catalyze the oxidation of .OH scavengers or promote the production of .OH, even at ratios of P-450/reductase approaching those found with intact microsomes and even in the presence of excess iron-EDTA, whereas ethanol is directly oxidized by P-450-PB, as are typical drug substrates. However, the P-450-PB-dependent oxidation of ethanol can be masked under conditions in which .OH production is increased.

Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis.

Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48-72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

Activation and detoxification of dinitropyrenes by cytosol and microsomes from Aroclor-pretreated rats in the Ames and umu assays.

1,3-, 1,6-, and 1,8-Dinitropyrene (1,3-, 1,6-, and 1,8-DNP) are direct-acting mutagens in that they do not require an exogenous source of enzymes for activation to mutagens in the Ames assay. However, the addition of mammalian S9 preparations, or the microsomal and cytosolic compartments comprising S9, modulate the mutagenic response of these DNPs. In this study, we compared the mutagenic response of these DNPs in the presence of cytosol and microsomal fractions from the liver of Aroclor-pretreated (AR) and control rats, in the Ames mutagenicity assay and umu gene induction assay. 1,3- and 1,8-DNP were deactivated to a greater extent by microsomes from AR-induced and control rats than was 1,6-DNP, in both the umu and Ames assays. In the Ames assay, S9 was more potent in deactivating the DNP than an equivalent concentration of microsomes from the same S9 preparation. Also, S9 from AR-pretreated rats deactivated the isomers to a greater extent than S9 from control rats. In contrast to the constant deactivation of all the isomers in the two assays catalyzed by microsomes and S9, the response with cytosol from AR-pretreated rats differed with respect to the three isomers in the Ames and umu assays. When cytosol from AR-treated rats was added, the mutagenicity of 1,3- and 1,6-DNP, but not 1,8-DNP, was significantly (P < 0.05) increased in the Ames assay while the mutagenicity of the three DNPs was increased in the umu assay. Also, a biphasic response was observed in the umu assay with 1,6- and 1,8-DNP, in that AR-cytosol enhanced the mutagenicity at low protein concentrations (5-50 micrograms protein/reaction) but abrogated the response at higher protein concentrations. The effect of cytosol from control rats depended on the isomer tested; 1,3-DNP was activated above the background level in both assays (nearly towfold) while 1,6-DNP and 1,8-DNP were only activated at low protein concentrations in the umu assay. In the Ames assay, cytosol from AR-pretreated rats did not alter the mutagenic response with 1,8-DNP, while control cytosol significantly (P < 0.05) deactivated 1,8-DNP at all substrate concentrations tested. In summary, this study showed that the mutagenicity of 1,3-DNP was similar in the two assays but the responses with 1,6- and 1,8-DNP differed in the two assays. These isomeric differences could be due to the varying metabolic pathways of the three DNPs as well as the detectable end points of the two assays.

Inhibition of rat heart mitochondrial electron transport in vitro: implications for the cardiotoxic action of allylamine or its primary metabolite, acrolein.

Allylamine (3-aminopropene) is a specific cardiac toxicant that causes aortic, valvular and myocardial lesions in many species. Myocardial necrosis can be observed 24 h after a single dose. Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein (2-propenal). Allylamine has been shown to bind to mitochondria from aorta and heart, suggesting that the subcellular site of injury is at or near the mitochondrion. The present investigation compared the effect of allylamine and its primary metabolite, acrolein, on electron transport and oxidative phosphorylation in mitochondria isolated from rat heart (RHM). Both compounds weakly inhibited mitochondrial electron transport with either the combination of glutamate, malate, and malonate (GMM, NADH-linked) or succinate as substrate. Comparisons of the slopes of concentration-effect regression (range of concentrations tested, 0.20-2.0 mM) lines showed acrolein to have significantly greater inhibitory effects than allylamine (range of concentrations tested, 0.22-6.4 mM) on GMM oxidation, while no significant difference in the abilities of the compounds to inhibit succinate oxidation were observed, indicating site preferences for inhibitory action. The addition of an uncoupling agent could not reverse inhibition with either substrate system. These results indicate that both the parent compound and its proposed metabolite primarily inhibit electron transport with little direct effect on the coupling mechanism. The State III EC50 (effective concentrations for 50% inhibition of control mitochondrial enzyme activities) for allylamine (2.29 mM with succinate as substrate and 1.22 mM with GMM) and acrolein (0.80 mM with succinate as substrate and 0.39 mM with GMM) are probably too great to invoke the direct action of either the parent compound or its oxidized metabolite on mitochondrial electron transport as a primary mechanism in the cardiotoxic action of allylamine.

Disodium tetrachloropalladate (Na2PdCl4), an inhibitor of rat liver mitochondrial electron transport.

The effects of Na2PdCl4 were studied on isolated rat liver mitochondrial electron transport and oxidative phosphorylation in vitro. Significant reductions in ADP-stimulated respiration were observed with increasing Na2PdCl4 concentrations with both succinate and NADH-linked substrate oxidations. Concentration necessary for half-maximal inhibition of oxygen uptake (EC50) for an NADH-linked substrate system was 18 muM while with succinate as substrate the EC50 was 15 muM. At 64 muM both systems were inhibited maximally at 60 and 80%, respectively. At concentrations of Na2PdCl4 sufficient to inhibit acceptor-stimulated oxygen uptake, there was a concomitant decrease in the rate of ADP phosphorylation as measured by proton absorption. Uncoupling agents had no effect on Na2PdCl4 inhibited mitochondria. Mg-ATPase activity and phosphate acceptor limited (State 4) respiratory activity were not stimulated by any Na2PdCl4 concentration used in these investigations. Data from these experiments indicate that Na2PdCl4 inhibits the mitochondrial respiratory chain in vitro.

Patterns of proliferative changes in crypts bordering colonic tumors: zonal histology and cell cycle marker expression.

Proliferative crypt changes have been noted in mucosa bordering colonic carcinomas, but their biological significance is disputed. We anticipated that zonal patterning of histological changes and cell cycle marker expression would provide clues to the pathogenesis of these border changes. 81 specimens were examined including carcinomas, adenomatours polyps, adenomas with early carcinoma, flat adenomas and aberrant crypt foci. The spatial distribution and frequency of micro-architectural features, and mucosal thickness were determined in a border domain of 150 300 sequential crypts/specimen. Immunocytochemical expression of Ki67 and p53 antigens in crypts also was semi-quantitatively examined. We found that in 100% of carcinomas two histologically abnormal zones (Proximate and Middle) separated tumor from normal mucosa. Differences in the feature frequency between zones were statistically significant (p<0.05). Both zones showed mild increases in crypt cell expression of Ki67, with a statistically significant relationship to zonal patterning (p<0.005). Weak expression of p53 only appeared in rare cells. Crypt elongation with mucosal thickening (1.9x normal, p<0.001) in the Proximate and Middle zones distinguished carcinomas from border changes in all benign lesions, except flat adenomas. Since this change occurs in all cases of carcinoma, there is no correlation with tumor stage or grade. Also in carcinomas, elaborate complexes of attached crypts (connected crypt structures) were characteristic of the Middle zone, so that proximate zone was always architecturally simpler. We conclude, that despite continuous carcinoma growth, the invaded border mucosa maintains a prototypical zonal organization of molecular and histological crypt changes This spatially organized reaction pattern is likely to reflect an interplay between regulated growth and destructive processes in response to advancing carcinoma. Compared to the edges of benign colonic tumors, the edges of carcinomas are distinctive and consistent enough to be diagnostically useful.

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment.

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.

Mutant frequency of lacI in transgenic mice following benzo[a]pyrene treatment and partial hepatectomy.

The Big Blue, transgenic mouse provides an in vivo mutation system that permits the study of pharmacodynamic parameters on mutant frequency (MF) following xenobiotic exposure. We have studied the effects of cellular proliferation on the frequency of mutations in the lacl transgene by evaluating the MF in the liver of male C57B1/6 Big Blue mice following treatment with benzo[a]pyrene (B[a]P) and a partial hepatectomy. Mice received either 40 mg/kg of B[a]P in corn oil or corn oil alone by i.p. injection on three consecutive days, followed by a partial hepatectomy on the fourth day. Three days later (i.e., 7 days following the initial B[a]P injection), the animals were sacrificed and the MF in the liver was compared to the MF observed in the liver of the same mouse at the time of hepatectomy. Induction of cytochrome P-450 1A (CYP1A) following B[a]P treatment was evident by Western blot analysis. The MF in untreated control animals was not significantly different at hepatectomy (4.7 +/- 0.8 x 10(-5)) and 3 days later, at sacrifice (3.0 +/- 0.4 x 10(-5)). Neither was the MF observed in the B[a]P-treated mice at the time of sacrifice (12.0 +/- 2.1 x 10(-5)) significantly different from the MF observed at the time of hepatectomy (10.6 +/- 5.3 x 10(-5)). However, B[a]P-treatment resulted in a 4.0-fold increase in MF at sacrifice which was significantly different (p < 0.05), when compared to the untreated controls. The B[a]P-treated mice at hepatectomy showed a modest 2.2-fold increase in MF which was not statistically significantly different from the untreated controls. In addition, both control and B[a]P-treated tissues gave sectored mutant plaques. The sectored plaque frequency (SPF) was significantly elevated (p < 0.05) in the B[a]P-treated mice at hepatectomy (4.2 +/- 1.0 x 10(-5)) and sacrifice (7.3 +/- 2.4 x 10(-5)) as compared to the respective frequency in the control mice at hepatectomy (1.9 +/- 0.7 x 10(-5)) and sacrifice (1.4 +/- 0.2 x 10(-5)). One explanation for this data is the persistence of the B[a]P adducts in the mouse genomic DNA that was packaged into the lambda phage, and ultimately fixed as mutations in Escherichia coli.