Doxorubicin-induced delayed-onset subclinical cardiotoxicity in mice.

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long-term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed-onset cardiotoxicity. The 6- or 9-mg/kg total cumulative doses resulted in a significant time-dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24-week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4- to 24-week period after treatment compared to SAL-treated counterparts. At 24 weeks after DOX treatment, a significant dose-related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose-related increase in the transcript level of Casp12 (SR-specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose-related elevation in the number of apoptotic cardiomyocytes compared to SAL-treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life.

Evaluating the Sub-Acute Toxicity of Formaldehyde Fumes in an In Vitro Human Airway Epithelial Tissue Model.

Formaldehyde (FA) is an irritating, highly reactive aldehyde that is widely regarded as an asthmagen. In addition to its use in industrial applications and being a product of combustion reaction and endogenous metabolism, FDA-regulated products may contain FA or release FA fumes that present toxicity risks for both patients and healthcare workers. Exposure to airborne FA is associated with nasal neoplastic lesions in both animals and humans. It is classified as a Group 1 carcinogen by International Agency for Research on Cancer (IARC) based on the increased incidence of cancer in animals and a known human carcinogen in the Report on Carcinogens by National Toxicology Program (NTP). Herein, we systematically evaluated the tissue responses to FA fumes in an in vitro human air-liquid-interface (ALI) airway tissue model. Cultures were exposed at the air interface to 7.5, 15, and 30 ppm of FA fumes 4 h per day for 5 consecutive days. Exposure to 30 ppm of FA induced sustained oxidative stress, along with functional changes in ciliated and goblet cells as well as possible squamous differentiation. Furthermore, secretion of the proinflammatory cytokines, IL-1ß, IL-2, IL-8, GM-CSF, TNF-a and IFN-γ, was induced by repeated exposures to FA fumes. Expression of MMP-1, MMP-3, MMP-7, MMP-10, MMP-12, and MMP-13 was downregulated at the end of the 5-day exposure. Although DNA-damage was not detected by the comet assay, FA exposures downregulated the DNA repair enzymes MGMT and FANCD2, suggesting its possible interference in the DNA repair capacity. Overall, a general concordance was observed between our in vitro responses to FA fume exposures and the reported in vivo toxicity of FA. Our findings provide further evidence supporting the application of the ALI airway system as a potential in vitro alternative for screening and evaluating the respiratory toxicity of inhaled substances.

Subacute Pulmonary Toxicity of Glutaraldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Glutaraldehyde (GA) has been cleared by the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration (FDA) as a high-level disinfectant for disinfecting heat-sensitive medical equipment in hospitals and healthcare facilities. Inhalation exposure to GA is known to cause respiratory irritation and sensitization in animals and humans. To reproduce some of the known in vivo effects elicited by GA, we used a liquid aerosol exposure system and evaluated the tissue responses in a human in vitro airway epithelial tissue model. The cultures were treated at the air interface with various concentrations of GA aerosols on five consecutive days and changes in tissue function and structure were evaluated at select timepoints during the treatment phase and after a 7-day recovery period. Exposure to GA aerosols caused oxidative stress, inhibition of ciliary beating frequency, aberrant mucin production, and disturbance of cytokine and matrix metalloproteinase secretion, as well as morphological transformation. Some effects, such as those on goblet cells and ciliated cells, persisted following the 7-day recovery period. Of note, the functional and structural disturbances observed in GA-treated cultures resemble those found in ortho-phthaldehyde (OPA)-treated cultures. Furthermore, our in vitro findings on GA toxicity partially and qualitatively mimicked those reported in the animal and human survey studies. Taken together, observations from this study demonstrate that the human air-liquid-interface (ALI) airway tissue model, integrated with an in vitro exposure system that simulates human inhalation exposure, could be used for in vitro-based human hazard identification and the risk characterization of aerosolized chemicals.

Toxicity of Ortho-phthalaldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Ortho-phthalaldehyde (OPA) is a chemical disinfectant used for the high-level sterilization of heat-sensitive medical instruments. Although OPA is considered a safer alternative to glutaraldehyde, no exposure limits have been established for respiratory exposures to ensure the safety of OPA sterilization and the safe use of OPA-treated medical instruments. In order to address data gaps in the toxicological profile of OPA, we treated human in vitro air-liquid-interface (ALI) airway cultures at the air interface with various concentrations of OPA aerosols for 10 consecutive days. Temporal tissue responses were evaluated at multiple time points during the treatment phase as well as 10 days following the last exposure. The disturbance of glutathione (GSH) homeostasis occurred as early as 20 min following the first exposure, while oxidative stress persisted throughout the treatment phase, as indicated by the sustained induction of heme oxygenase-1 (HMOX-1) expression. Repeated exposures to OPA aerosols resulted in both functional and structural changes, including the inhibition of ciliary beating frequency, aberrant mucin production, decreases in airway secretory cells, and tissue morphological changes. While OPA-induced oxidative stress recovered to control levels after a 10 day recovery period, functional and structural alterations caused by the high concentration of OPA aerosols failed to fully recover over the observation period. These findings indicate that aerosolized OPA induces both transient and relatively persistent functional and structural abnormalities in ALI cultures under the conditions of the current study.

Characterization of the variability in the extent of nonalcoholic fatty liver induced by a high-fat diet in the genetically diverse Collaborative Cross mouse model.

Interindividual variability and sexual dimorphisms in the development of nonalcoholic fatty liver disease (NAFLD) are still poorly understood. In the present study, male and female strains of Collaborative Cross (CC) mice were fed a high-fat and high-sucrose (HF/HS) diet or a control diet for 12 weeks to investigate interindividual- and sex-specific variations in the development of NAFLD. The severity of liver steatosis varied between sexes and individual strains and was accompanied by an elevation of serum markers of insulin resistance, including increases in total cholesterol, low-density lipoproteins, high-density lipoproteins, phospholipids, and glucose. The development of NAFLD was associated with overexpression of the critical fatty acid uptake and de novo lipogenesis genes Pparg, Mogat1, Cd36, Acaab1, Fabp2, and Gdf15 in male and female mice. The expression of Pparg, Mogat1, and Cd36 was positively correlated with liver triglycerides in male mice, and Mogat1 and Cd36 expression were positively correlated with liver triglycerides in female mice. Our results indicate the value of CC mice in combination with HF/HS diet-induced alterations as an approach to study the susceptibility and interindividual variabilities in the pathogenesis of nonalcoholic fatty liver and early nonalcoholic steatohepatitis at the population level, uncovering of susceptible and resistant cohorts, and identifying sex-specific molecular determinants of disease susceptibility.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air-liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1 week (a total of 3 days, 40 min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Sex-related differential susceptibility to doxorubicin-induced cardiotoxicity in B6C3F1 mice.

Sex is a risk factor for development of cardiotoxicity, induced by the anti-cancer drug, doxorubicin (DOX), in humans. To explore potential mechanisms underlying differential susceptibility to DOX between sexes, 8-week old male and female B6C3F1 mice were dosed with 3mg/kg body weight DOX or an equivalent volume of saline via tail vein once a week for 6, 7, 8, and 9 consecutive weeks, resulting in 18, 21, 24, and 27mg/kg cumulative DOX doses, respectively. At necropsy, one week after each consecutive final dose, the extent of myocardial injury was greater in male mice compared to females as indicated by higher plasma concentrations of cardiac troponin T at all cumulative DOX doses with statistically significant differences between sexes at the 21 and 24mg/kg cumulative doses. A greater susceptibility to DOX in male mice was further confirmed by the presence of cytoplasmic vacuolization in cardiomyocytes, with left atrium being more vulnerable to DOX cardiotoxicity. The number of TUNEL-positive cardiomyocytes was mostly higher in DOX-treated male mice compared to female counterparts, showing a statistically significant sex-related difference only in left atrium at 21mg/kg cumulative dose. DOX-treated male mice also had an increased number of γ-H2A.X-positive (measure of DNA double-strand breaks) cardiomyocytes compared to female counterparts with a significant sex effect in the ventricle at 27mg/kg cumulative dose and right atrium at 21 and 27mg/kg cumulative doses. This newly established mouse model provides a means to identify biomarkers and access potential mechanisms underlying sex-related differences in DOX-induced cardiotoxicity.

Hepatic Overexpression of Annexin A1 and A2 in Thioacetamide-Primed Mice Protects Them Against Acetaminophen-Induced Liver Failure and Death.

Compensatory tissue repair (CTR) in thioacetamide (TA)-primed rats protects them against acetaminophen (APAP)-induced lethality. This study was aimed at investigating the mechanisms of CTR-mediated heteroprotection in mice. Male Swiss Webster mice received a priming dose of TA (40 mg/kg body weight [BW] in 10 mL distilled water [DW]/kg BW, intraperitoneally [IP]). Thioacetamide-induced liver injury, CTR, and expression of annexin A1 and A2 (ANX1 and ANX2), the endogenous inhibitors of the death protein secretory phospholipase A2 (sPLA2), were measured over a time course of 84 hours after TA priming. Both centrilobular necrosis and CTR peaked at 36 hours after TA priming as indicated by significantly increased plasma alanine transaminase (ALT) and aspartate transaminase (AST) activities, liver histology, and proliferating cell nuclear antigen immunostaining. Thioacetamide priming resulted in the overexpression of ANX1 and ANX2 at 36 to 84 hours and 12 to 60 hours, respectively. A lethal dose of APAP (600 mg/kg BW in 10 mL 0.45% NaCl/kg BW, IP) was given at 12, 24, or 36 hours after TA-priming. Thioacetamide priming did not affect the rise in plasma ALT, AST, sPLA2, and arachidonic acid levels seen at 2 hours after the APAP overdose. Neither these biochemical parameters nor histology suggested any escalation of hepatic injury at later time points (12 and 24 hours after APAP overdose), consistent with 100% survival of the TA + APAP-treated mice compared to DW + APAP-treated mice, which had 100% mortality. Inhibition of ANX1 and ANX2 biosynthesis using cycloheximide (40 mg/kg BW in 5 mL DW/kg BW, IP) abolished this heteroprotection. Our data indicate that hepatic overexpression of ANX1 and ANX2 inhibits APAP-induced expansion of liver injury.

Tight junction disruption by cadmium in an in vitro human airway tissue model.

BACKGROUND: The cadmium (Cd) present in air pollutants and cigarette smoke has the potential of causing multiple adverse health outcomes involving damage to pulmonary and cardiovascular tissue. Injury to pulmonary epithelium may include alterations in tight junction (TJ) integrity, resulting in impaired epithelial barrier function and enhanced penetration of chemicals and biomolecules. Herein, we investigated mechanisms involved in the disruption of TJ integrity by Cd exposure using an in vitro human air-liquid-interface (ALI) airway tissue model derived from normal primary human bronchial epithelial cells. METHODS: ALI cultures were exposed to noncytotoxic doses of CdCl2 basolaterally and TJ integrity was measured by Trans-Epithelial Electrical Resistance (TEER) and immunofluorescence staining with TJ markers. PCR array analysis was used to identify genes involved with TJ collapse. To explore the involvement of kinase signaling pathways, cultures were treated with CdCl2 in the presence of kinase inhibitors specific for cellular Src or Protein Kinase C (PKC). RESULTS: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining. CdCl2 exposure altered the expression of several groups of genes encoding proteins involved in TJ homeostasis. In particular, down-regulation of select junction-interacting proteins suggested that a possible mechanism for Cd toxicity involves disruption of the peripheral junctional complexes implicated in connecting membrane-bound TJ components to the actin cytoskeleton. Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins. CONCLUSIONS: Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

Early biomarkers of doxorubicin-induced heart injury in a mouse model.

Cardiac troponins, which are used as myocardial injury markers, are released in plasma only after tissue damage has occurred. Therefore, there is a need for identification of biomarkers of earlier events in cardiac injury to limit the extent of damage. To accomplish this, expression profiling of 1179 unique microRNAs (miRNAs) was performed in a chronic cardiotoxicity mouse model developed in our laboratory. Male B6C3F1 mice were injected intravenously with 3mg/kg doxorubicin (DOX; an anti-cancer drug), or saline once a week for 2, 3, 4, 6, and 8weeks, resulting in cumulative DOX doses of 6, 9, 12, 18, and 24mg/kg, respectively. Mice were euthanized a week after the last dose. Cardiac injury was evidenced in mice exposed to 18mg/kg and higher cumulative DOX dose whereas examination of hearts by light microscopy revealed cardiac lesions at 24mg/kg DOX. Also, 24 miRNAs were differentially expressed in mouse hearts, with the expression of 1, 1, 2, 8, and 21 miRNAs altered at 6, 9, 12, 18, and 24mg/kg DOX, respectively. A pro-apoptotic miR-34a was the only miRNA that was up-regulated at all cumulative DOX doses and showed a significant dose-related response. Up-regulation of miR-34a at 6mg/kg DOX may suggest apoptosis as an early molecular change in the hearts of DOX-treated mice. At 12mg/kg DOX, up-regulation of miR-34a was associated with down-regulation of hypertrophy-related miR-150; changes observed before cardiac injury. These findings may lead to the development of biomarkers of earlier events in DOX-induced cardiotoxicity that occur before the release of cardiac troponins.

Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury.

Dysregulation of one-carbon metabolism-related metabolic processes is a major contributor to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). It is well established that genetic and gender-specific variations in one-carbon metabolism contribute to the vulnerability to NAFLD in humans. To examine the role of one-carbon metabolism dysregulation in the pathogenesis and individual susceptibility to NAFLD, we used a "population-based" mouse model where male mice from 7 inbred were fed a choline- and folate-deficient (CFD) diet for 12 wk. Strain-dependent down-regulation of several key one-carbon metabolism genes, including methionine adenosyltransferase 1α (Mat1a), cystathionine-ß-synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosyl-homocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), was observed. These changes were strongly associated with interstrain variability in liver injury (steatosis, necrosis, inflammation, and activation of fibrogenesis) and hyperhomocysteinemia. Mechanistically, the decreased expression of Mat1a, Ahcy, and Mthfd1 was linked to a reduced level and promoter binding of transcription factor CCAAT/enhancer binding protein ß (CEBPß), which directly regulates their transcription. The strain specificity of diet-induced dysregulation of one-carbon metabolism suggests that interstrain variation in the regulation of one-carbon metabolism may contribute to the differential vulnerability to NFLD and that correcting the imbalance may be considered as preventive and treatment strategies for NAFLD.

Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations.

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Mouse liver protein sulfhydryl depletion after acetaminophen exposure.

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

Interstrain differences in liver injury and one-carbon metabolism in alcohol-fed mice.

UNLABELLED: Alcoholic liver injury is a major public health issue worldwide. Even though the major mechanisms of this disease have been established over the past decades, little is known about genetic susceptibility factors that may predispose individuals who abuse alcoholic beverages to liver damage and subsequent pathological conditions. We hypothesized that a panel of genetically diverse mouse strains may be used to examine the role of endoplasmic reticulum (ER) stress and one-carbon metabolism in the mechanism of interindividual variability in alcoholic liver injury. We administered alcohol (up to 27 mg/kg/d) in a high-fat diet using an intragastric intubation model for 28 days to male mice from 14 inbred strains (129S1/SvImJ, AKR/J, BALB/cJ, BALB/cByJ, BTBR T+tf/J, C3H/HeJ, C57BL/10J, DBA/2J, FVB/NJ, KK/HIJ, MOLF/EiJ, NZW/LacJ, PWD/PhJ, and WSB/EiJ). Profound interstrain differences (more than 3-fold) in alcohol-induced steatohepatitis were observed among the strains in spite of consistently high levels of urine alcohol that were monitored throughout the study. We found that ER stress genes were induced only in strains with the most liver injury. Liver glutathione and methyl donor levels were affected in all strains, albeit to a different degree. The most pronounced effects that were closely associated with the degree of liver injury were hyperhomocysteinemia and strain-dependent differences in expression patterns of one-carbon metabolism-related genes. CONCLUSION: Our data demonstrate that strain differences in alcohol-induced liver injury and steatosis are striking and independent of alcohol exposure and the most severely affected strains exhibit major differences in the expression of ER stress markers and genes of one-carbon metabolism.

Changes in mouse liver protein glutathionylation after acetaminophen exposure.

The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses.

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform.

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with con-current cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

The role of epigenetic events in genotoxic hepatocarcinogenesis induced by 2-acetylaminofluorene.

It is well established that genotoxic reactivity of chemical carcinogens or their metabolites is a critical event in the initiation of tumorigenesis. However, the underlying mechanisms of events following initiation are less well understood, and with respect to genotoxic liver carcinogenesis, it is largely unknown how the initiated cells progress to form preneoplastic hepatic foci. In the present study, we investigated the underlying events associated with tumor-promoting activity of 2-acetylaminofluorene (2-AAF), a powerful complete genotoxic rat liver carcinogen. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 24 weeks, and the status of cytosine DNA methylation, histone methylation, and microRNA expression was determined in the livers of control and 2-AAF-fed rats. The results demonstrate that stages of multistage carcinogenesis following the initiation are driven primarily by carcinogen-induced epigenetic alterations. This was evidenced by altered global histone lysine methylation patterns, increased histone H3 lysine 9 and histone H3 lysine 27 trimethylation in the promoter regions of Rassf1a, p16(INK4a), Socs1, Cdh1, and Cx26 tumor suppressor genes, early Rassf1a and p16(INK4a) promoter CpG island hypermethylation, and altered microRNA expression in preneoplastic livers of rats exposed to 2-AAF. These changes were accompanied by dysregulation of the balance between cell proliferation and apoptosis, a fundamental pro-tumorigenic event in hepatocarcinogenesis. These results signify the fundamental role of epigenetic alterations in genotoxic liver carcinogenesis.