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The comparatively high cost of laboratory detection methods for Clostridium difficile infection (CDI) coupled to a low prevalence rate has resulted in testing algorithms that use cheaper and relatively sensitive screening methods, followed by more specific confirmatory methods. The aim of this prospectively-conducted study from two centres in the UK, and one in the Republic of Ireland was to determine the efficacy of the EntericBio ® realtime C. difficile Assay (EBCD) for the detection of toxigenic C. difficile in stool samples. The EBCD was compared to the in-use testing methods for Clostridium difficile (CD) detection in each centre. In the two UK centres, the EBCD was compared to the C.diff Quik Chek Complete ® kit (Techlab), and discrepancies were tested further using The Xpert ® C. difficile PCR assay (Cepheid) and PCR ribotyping after cultivation using the spore culture method, respectively. In the Irish centre, EBCD comparison was to an algorithm of C. DIFF CHEK™-60 test (Techlab) for screening followed by C. difficile Premier ™ Toxins A&B assay (Meridian Bioscience ® ) in the case of positive results; discrepancies were tested using the Xpert ® C. difficile PCR assay. In a retrospective analysis of data, a total of 947 stool samples were tested, of which eight (0.8%) proved inhibitory to the EBCD assay. Of the 939 valid tests conducted, reported sensitivities of the EBCD were 94.7%, 100% and 97.9%, respectively; specificities were 99.6%, 100% and 100%, respectively; positive predictive values were 94.7%, 100% and 100%, respectively, and negative predictive values were 99.6%, 100% and 99.8%, respectively. The CD positivity rates in the current study ranged between 6.6% and 8.2%., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as acute or chronic toxicities. Alternately, suppression of apoptosis through, for example, interference in cell signaling can result in pathological accumulation of aberrant cells and diseases such as tumors. Investigations into the mechanisms underlying apoptosis have extended into many areas, driven by increasingly sophisticated instrumental and molecular biology techniques. This symposium summary explores related aspects of apoptosis, including control of cell population size and function, specific gene activity and regulation, chromatin condensation and scaffold detachment, oxidative stress-induced cell proliferation versus death by apoptosis or necrosis, and hepatotoxicant-induced apoptosis versus necrosis. Insights into the mechanisms governing apoptosis and increasing appreciation of the relevance of apoptotic cell death are redirecting research in toxicology and carcinogenesis and are yielding novel therapeutic approaches for the control of toxicity, disease, and ultimately perhaps senescence.

Hepatocellular necrosis occurs under a wide range of pathological conditions. In most cases, toxic cell death takes place over a finite span of time, delayed from the point of initial injury and accompanied by homeostatic counterresponses that are varied and complex. The present strategies for discovering critical steps in cell death recognize that (1) different toxins produce similar morphologic changes that precede killing in widely varied cell types, and that (2) lethal events are likely to involve one or more compartmentalized functions that are common to most cells. Investigations of the plasma membrane, endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus have greatly advanced our understanding of acute hepatocellular necrosis. This report examines each compartment but emphasizes molecular changes in the nucleus which may explain cell death caused by alkylating hepatotoxicants. Accumulating knowledge about two distinct modes of cell death, necrosis and apoptosis, indicates that loss of Ca2+ regulation and subsequent damage to DNA may be critical steps in lethal damage to liver cells by toxic chemicals.

The purpose of these studies was to evaluate the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on intracellular free Ca2+ and DNA fragmentation in lymphoid cells obtained from the spleen, thymus, and Peyer's patches (PPs) of female B6C3F1 mice. Previous studies in our laboratories have shown that DMBA is cytotoxic to these lymphoid organs and that calcium homeostasis may be impaired following DMBA treatment. The results of the present studies show that a daily oral 14-day exposure of mice to DMBA produced a dose-dependent decrease in the number of viable cells recovered from the spleen, PPs, and thymus. Intracellular levels of Ca2+ were elevated in the spleen and PPs of mice receiving 140 mg/kg of DMBA. Extensive DNA fragmentation was detected in cells obtained from the spleen and PPs, as well as from the thymus. The thymus and PPs demonstrated DNA fragmentation at significantly lower doses of DMBA (42 mg/kg) than did the spleen (140 mg/kg). While cells obtained from the thymus did not demonstrate an elevation in Ca2+ produced by DMBA, in vitro exposure of isolated thymocytes to 3-30 microM DMBA for 4 hr produced significant elevation of intracellular Ca2+. A "ladder-like" pattern of DNA fragmentation was seen by agarose gel electrophoresis of DNA obtained from thymus cells treated with DMBA in vitro, suggesting DNA degradation by endonucleases. Collectively, these studies suggest that DMBA produces lymphotoxicity through an apoptosis-like mechanism involving fragmentation of genomic DNA by Ca(2+)-activated enzymes.

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.

Hepatotoxic doses of acetaminophen cause widespread alkylation of liver and early loss of cytosolic Ca2+ regulation. Although the precise location and target of lethal alkylation are not known, Ca2+ accumulation is viewed as a possible link between cell alkylation and cell death. We have recently shown that Ca2+ accumulates in the nucleus and that DNA fragments in vivo before the development of acetaminophen-induced necrosis in mice. The present study examined cultured hepatocytes for nuclear damage and its association with cell death in vitro. Positive results would argue for two key points. (1) Nonparenchymal cell damage does not explain DNA fragmentation induced by acetaminophen in vivo. (2) A chemical that causes necrosis can produce DNA damage considered characteristic of apoptosis. Hepatocytes from NIH Swiss mice were isolated by collagenase perfusion, cultured in Williams' E medium for 24 hr, and exposed to acetaminophen. Cytotoxicity was assessed by lactate dehydrogenase leakage and release of [3H]adenine from a prelabeled nucleotide pool. Genomic DNA fragmentation was assessed quantitatively by colorimetric analysis and qualitatively by agarose gel electrophoresis. Acetaminophen caused DNA damage from 1-4 hr onward and produced significant release of lactate dehydrogenase and [3H]adenine nucleotides at later times. Agarose gel electrophoresis revealed a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. Cytotoxicity correlated with nuclear Ca2+ accumulation (r greater than 0.895, p less than 0.05) and with percentage DNA fragmentation (r greater than 0.835, p less than 0.05). Nuclear changes in vitro generally reproduced those observed in vivo. Collectively, these findings demonstrate that nuclear Ca2+ accumulation and DNA fragmentation appear as early events that correlate directly with later cytotoxicity. These changes may contribute to acetaminophen-induced injury leading to cell death in vitro and necrosis in vivo.

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca2+ action by evaluating whether acetaminophen raises Ca2+ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca2+ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by greater than 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT greater than 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca2+ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.

Administration of 1200 mg/kg N-acetyl-L-acetyl-L-cysteine to mice apparently increased the rate of glutathione synthesis resulting in prolonged elevation of the hepatic glutathione pool. At 3 hr, peak glutathione concentrations nearly doubled. The fraction of glutathione in the oxidized state remained at normally low values over this period. The L isomer also significantly increased plasma and urinary concentrations of inorganic sulfate. Plasma concentrations nearly doubled at their peak and urinary excretion over 24 hr rose some threefold above control. Consistent with the known stereoselectivity of many biological processes, the unnatural D isomer of N-acetylcysteine failed to increase hepatic glutathione. Liver concentrations remained similar to control suggesting that the D isomer was unable to increase the rate of glutathione synthesis. The D isomer further differed from its L enantiomer in failing to increase the plasma concentration and the urinary excretion of inorganic sulfate. Congruent with these observations, much more N-acetyl-D-cysteine (47% of dose) was recovered unchanged in 24 hr urine than N-acetyl-L-cysteine (6.1% of dose). These findings are of toxicologic interest because they identify the N-acetylcysteine stereoisomers as a pair of agents that may be useful in separating biologic effects of sulfhydryls that are related primarily to their physical properties (i.e. reduction, radical scavenging) from those that accrue from their ability to increase glutathione and sulfate availability in vivo.

Effects of the diuretic drug furosemide were examined in obese animals to evaluate the hypothesis that organ damage by reactive drug metabolites may be potentiated by this disease. Obese overfed Sprague-Dawley rats that were treated ip with 450 mg/kg furosemide on the basis of total body mass suffered a 58% mortality rate over 24 h. This contrasted with 0% mortality in animals of normal body mass. On the basis of median histopathology scores, organ necrosis was judged to be greater in the liver (2+) and kidneys (1+) of obese rats than in the liver (1+) and kidneys (less than 1+) of normal controls (p less than 0.05). Obese animals demonstrated a fourfold rise in fat mass over controls. The low solubility of furosemide in lipid makes it probable that aggravated drug toxicity in obese rats dosed to total body mass resulted in part from elevated furosemide concentrations in lean body mass. In a subsequent study designed to minimize this possibility, furosemide was administered on the basis of fat-free body mass to equalize initial drug exposure in obese and control rats. Even with this downward dosage adjustment, obese animals suffered increased hepatic necrosis (median score of 2+ versus 0 in treated controls), greater impairment of renal function (plasma creatinine concentration of 2.41 mg/dl versus 0.96 mg/dl in treated controls), and more extensive enzymuria (enzyme excretion 175-300% more elevated than in treated controls). In conclusion, obese rats appear to be at increased risk of furosemide-induced liver and kidney injury due to at least two factors: (1) increased exposure of target organs in lean body mass to furosemide when the dosing of this poorly lipophilic drug was based on total body mass, and (2) increased susceptibility of target organs in lean body mass to furosemide injury when dosing was adjusted downward to reflect fat-free body mass and to equalize initial drug exposure.