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Toxicological Sciences

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The field of environmental research has benefited greatly from the concept of biomarkers, which originally expanded our thinking by opening the "black box" between environmental exposures and manifestations of disease and dysfunction in exposed populations, as laid out in a highly influential article published in 1987 by an expert committee convened by the National Research Council. Advances in biomedical research now challenge us to revise this concept to include the microbiome as a critical stage in the progression from exposure to outcome. Incorporating the microbiome into the basic 1987 model can spur new advances and understanding in environmental health. The human microbiome as a whole comprises the majority of cells and genes of the super-organism (host and microbiome). Site-specific microbiomes are the first to encounter xenobiotics, prior to absorption across gut, skin, or respiratory system. A growing literature indicates that these microbial communities may participate in biotransformation and thus constitute a compartment to add to the original biomarker schematic. In addition, these microbiomes interact with the "niche" in which they are located and thus transduce responses to and from the host organism. Incorporating the microbiome into the environmental health paradigm will enlarge our concepts of susceptibility as well as the interactions between xenobiotics and other factors that influence the status and function of these barrier systems. This article reviews the complexities of host:microbiome responses to xenobiotics in terms of redefining toxicokinetics and susceptibility. Our challenge is to consider these multiple interactions between and within the microbiome, the immune system, and other systems of the host in terms of exposure to exogenous agents, including environmental toxicants.

Furan is a heterocyclic organic compound produced in the chemical manufacturing industry and also found in a broad range of food products, including infant formulas and baby foods. Previous reports have indicated that the adverse biological effects of furan, including its liver tumorigenicity, may be associated with epigenetic abnormalities. In the present study, we investigated the persistence of epigenetic alterations in rat liver. Male F344 rats were treated by gavage 5 days per week with 8 mg furan/kg body weight (bw)/day for 90 days. After the last treatment, rats were divided randomly into 4 groups; 1 group of rats was sacrificed 24 h after the last treatment, whereas other groups were maintained without further furan treatment for an additional 90, 180, or 360 days. Treatment with furan for 90 days resulted in alterations in histone lysine methylation and acetylation, induction of base-excision DNA repair genes, suggesting oxidative damage to DNA, and changes in the gene expression in the livers. A majority of these furan-induced molecular changes was transient and disappeared after the cessation of furan treatment. In contrast, histone H3 lysine 9 and H3 lysine 56 showed a sustained and time-depended decrease in acetylation, which was associated with formation of heterochromatin and altered gene expression. These results indicate that furan-induced adverse effects may be mechanistically related to sustained changes in histone lysine acetylation that compromise the ability of cells to maintain and control properly the expression of genetic information.

Functional changes to cardiomyocytes are a common cause of attrition in preclinical and clinical drug development. Current approaches to assess cardiomyocyte contractility in vitro are limited to low-throughput methods not amenable to early drug discovery. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) were used to assess their suitability to detect drug-induced changes in cardiomyocyte contraction. Application of field stimulation and measurement of cardiac contraction (IonOptix edge detection) and Ca2+ transients confirmed hiPS-CMs to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a live cell, fast kinetic fluorescent assay with a Ca2+ sensitive dye to test 31 inotropic and 20 non-inotropic compounds in vivo, we report that hiPS-CMs provide a high-throughput experimental model to detect changes in cardiomyocyte contraction that is applicable to early drug discovery with a sensitivity and specificity of 87% and 70%, respectively. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with throughput amenable to influencing chemical design in drug discovery. Measurement of multiple parameters of the Ca2+ transient in addition to the number of Ca2+ transients offered no insight into the mechanism of cardiomyocyte contraction.

Cumulative fluoride exposure has adverse influences on children’s intelligence quotient (IQ). In addition, catechol-O-methyltransferase (COMT) gene Val158Met polymorphism (rs4680) is associated with cognitive performance. This study aimed to evaluate the associations of COMT polymorphism and alterations of protein profiles with children’s intelligence in endemic fluorosis area. We recruited 180 schoolchildren (10–12 years old) from high fluoride exposure (1.40 mg/l) and control areas (0.63 mg/l) in Tianjin City, China. The children’s IQ, fluoride contents in drinking water (W-F), serum (S-F), and urine (U-F); serum thyroid hormone levels, COMT Val158Met polymorphism, and plasma proteomic profiling were determined. Significant high levels of W-F, S-F, U-F, along with poor IQ scores were observed in the high fluoride exposure group compared with those in control (all P < 0.05). S-F and U-F were inversely related with IQ (rs = –0.47, P < 0.01; rs = –0.45, P = 0.002). Importantly, higher fluoride exposure was associated with steeper cognitive decline among children with the reference allele Val compared with those homozygous or heterozygous for the variant allele Met (95% CI, –16.80 to 2.55; P interaction < 0.01). Additionally, 5 up-regulated protein spots related to cell immunity and metabolism were detected in children with high fluoride exposure compared with the control. In conclusion, fluoride exposure was adversely associated with children’s intelligence, whereas the COMT polymorphism may increase the susceptibility to the deficits in IQ due to fluoride exposure. Moreover, the proteomic analysis can provide certain basis for identifying the early biological markers of fluorosis among children.

Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity.

Most toxicological testing focuses on defining concentration/dose-response relationships, with little consideration to temporal aspects of the expression of chemical effects. Yet, both dose and time are critical to evaluating potential risks. Our lab has generated an extensive amount of linked dose-response and time-course data for eight model endocrine-disrupting chemicals (EDCs) using adult fathead minnows (Pimephales promelas). Herein we conduct a meta-analysis of this novel dataset, with an emphasis on defining interactions between dose and time on several molecular and biochemical endpoints indicative of endocrine function, both during chemical exposure and recovery. Direct effects of the EDCs were very rapid, often occurring within 24 h or less. Recovery after removal of the chemical stressors was similarly rapid. All experiments provided evidence of system compensation. For example, measures of endocrine perturbation early in an experiment, especially in low-dose treatments, often recovered to control values while a chemical exposure was ongoing. In experiments with several of the EDCs, shortly after cessation of the chemical exposure there was an "overshoot" behavior, in which different measures of endocrine function exceeded control values. Overall, when an endpoint was measured was as an important determinant in identifying a chemical as endocrine-active, as the dose to which the animal was exposed. The compensatory responses during exposure and early recovery after removal of the chemical stressor also produced examples of nonmonotonic dose-response relationships. The types of interactions between time and dose observed in these studies have a number of important implications for screening, testing, and monitoring programs for EDCs.

Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na+ and/or Cl in the extracellular space and may be related to an influx of Na+ and/or a shift in Cl, which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca2+ moderately contributed to the swelling because benzamil (10 µM), a 3Na+/Ca2+ exchange (NCX) inhibitor, and Ca2+-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca2+ activity [Ca2+]i. This increase was dependent on the presence of Na+ and Ca2+ in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca2+ homeostasis to influence the toxin-induced respiratory arrest.

Exposure to Staphylococcal enterotoxin B (SEB) causes food poisoning, acute inflammatory lung injury, toxic shock syndrome, and often death. In this study, we investigated whether microRNA (miRNA) play a role in regulating SEB-driven inflammation in the lungs. Exposure to SEB caused immune cell infiltration, robust cytokine and chemokine production, compromised lung function, and 100% mortality in mice. We assessed miRNA and mRNA expression in lung infiltrating mononuclear cells following exposure to SEB and found 89 miRNA that were dysregulated (>2-fold) compared with vehicle controls. In silico analysis revealed that the miRNA exhibited biological functions pertaining to cell death and survival, cellular proliferation, and cell cycle progression. Through the use of q-RT PCR, we validated 9 specific miRNA (miR-155, miR-132, miR-31, miR-222, miR-20b, miR-34a, miR-192, miR-193*, and let-7e) and observed that they were predicted to bind the 3'-UTR of a number of genes that were either involved in the stringent regulation of inflammation (Smad3, Tgfb, Runx1, and Foxo3) or those that contributed to its exacerbation (Stat3, Ptgs2, Ccnd1, Ccne1, NfB, and Tbx21). Further, by increasing or decreasing the levels of miR-132 (a miRNA highly induced by SEB), we noted the corresponding decrease or increase in the levels of its predicted target FOXO3. As a result of FOXO3 suppression by miR-132, we saw increase in Ifn-, Ccnd, and Ccne1. Taken together, our data support the role for miRNA in actively participating and orchestrating SEB-mediated inflammation in the lungs and provide several therapeutic targets for the treatment of SEB-driven toxicity via the modulation of miRNA.

Although many countries banned of its usage, carbofuran (CF) is still one of the most commonly used carbamate derivative insecticides against insects and nematodes in agriculture and household, threatening the human and animal health by contaminating air, water, and food. Our goal was to evaluate the potential toxic effects of CF on mammalian oocytes besides mitotic cells. Caspase-dependent apoptotic pathway was assessed by immunofluorescence and western blot techniques. Alterations in the meiotic spindle formation after CF exposure throughout the in vitro maturation of mice oocyte-cumulus complexes (COCs) were analyzed by using a 3D confocal laser microscope. Maturation efficiency and kinetics were assessed by direct observation of the COCs. Results indicated that the number of TUNEL-positive cells increased in CF-exposed groups, particularly higher doses (>250 µM) in a dose-dependent fashion. The ratio of anticleaved caspase-3 labeled cells in those groups positively correlated with TUNEL-positivity. Western blot analysis confirmed a significant increase in active caspase-3 activity. CF caused a dose-dependent accumulation of oocytes at prometaphase-I (PM-I) of meiosis. Partial loss of spindle microtubules (MTs) was noted, which consequently gave rise to a diamond shape spindle. Aberrant pericentrin foci were noted particularly in PM-I and metaphase-I (M-I) stages. Conclusively, CF (1) induces programmed cell death in a dose-dependent manner, and (2) alters spindle morphology most likely through a mechanism that interacts with MT assembly and/or disorientation of pericentriolar proteins. Overall, data suggest that CF could give rise to aneuploidy or cell death in higher doses, therefore reduce fertilization and implantation rates.

Orotic acid (OA) is an intermediate of pyrimidine nucleotide biosynthesis. Hereditary deficiencies in some enzymes associated with pyrimidine synthesis or the urea cycle induce OA accumulation, resulting in orotic aciduria. A link between patients with orotic aciduria and hypertension has been reported; however, the molecular mechanisms remain elusive. In this study, to elucidate the role of OA in vascular insulin resistance, we investigated whether OA induced endothelial dysfunction and hypertension. OA inhibited insulin- or metformin-stimulated nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation in human umbilical vein endothelial cells. A decreased insulin response by OA was mediated by impairment of the insulin-stimulated phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway in cells overexpressing the p110-PI3K catalytic subunit. Impaired effects of metformin on eNOS phosphorylation and NO production were reversed in cells transfected with constitutively active AMP-activated protein kinase. Moreover, experimental induction of orotic aciduria in rats caused insulin resistance, measured as a 125% increase in the homeostasis model assessment, and hypertension, measured as a 25% increase in systolic blood pressure. OA increased the plasma concentration of endothelin-1 by 201% and significantly inhibited insulin- or metformin-induced vasodilation. A compromised insulin or metformin response on the Akt/eNOS and AMP-activated protein kinase/eNOS pathway was observed in aortic rings of OA-fed rats. Taken together, we showed that OA induces endothelial dysfunction by contributing to vascular and systemic insulin resistance that affects insulin- or metformin-induced NO production, leading to the development of hypertension.

Developmental manganese (Mn) exposure is associated with motor dysfunction in children and animal models, but little is known about the underlying neurochemical mechanisms or the potential for amelioration by pharmacotherapy. We investigated whether methylphenidate (MPH) alleviates fine motor dysfunction due to chronic postnatal Mn exposure, and whether Mn exposure impairs brain extracellular dopamine (DA) and norepinephrine (NE) in the prefrontal cortex (PFC) and striatum in adult animals. Rats were orally exposed to 0 or 50 mg Mn/kg/day from postnatal day 1 until the end of the study (PND 145). The staircase test was used to assess skilled forelimb function. Oral MPH (2.5 mg/kg/day) was administered daily 1 h before staircase testing for 16 days. DA and NE levels were measured by dual probe microdialysis. Results show that Mn exposure impaired reaching and grasping skills and the evoked release of DA and NE in the PFC and striatum of adult rats. Importantly, oral MPH treatment fully alleviated the fine motor deficits in the Mn-exposed animals, but did not affect forelimb skills of control rats not exposed to Mn. These results suggest that catecholaminergic hypofunctioning in the PFC and striatum may underlie the Mn-induced fine motor dysfunction, and that oral MPH pharmacotherapy is an effective treatment approach for alleviating this dysfunction in adult animals. The therapeutic potential of MPH for the treatment of motor dysfunction in Mn-exposed children and adults appears promising pending further characterization of MPH efficacy in other functional areas (eg, attention) believed to be affected by developmental Mn exposure.

Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells.

Chronic exposure to inorganic arsenic (iAs) has been linked to an increased risk of diabetes, yet the specific disease phenotype and underlying mechanisms are poorly understood. In the present study we set out to identify iAs exposure-associated metabolites with altered abundance in nondiabetic and diabetic individuals in an effort to understand the relationship between exposure, metabolomic response, and disease status. A nested study design was used to profile metabolomic shifts in urine and plasma collected from 90 diabetic and 86 nondiabetic individuals matched for varying iAs concentrations in drinking water, body mass index, age, and sex. Diabetes diagnosis was based on measures of fasting plasma glucose and 2-h blood glucose. Multivariable models were used to identify metabolites with altered abundance associated with iAs exposure among diabetic and nondiabetic individuals. A total of 132 metabolites were identified to shift in urine or plasma in response to iAs exposure characterized by the sum of iAs metabolites in urine (U-tAs). Although many metabolites were altered in both diabetic and nondiabetic 35 subjects, diabetic individuals displayed a unique response to iAs exposure with 59 altered metabolites including those that play a role in tricarboxylic acid cycle and amino acid metabolism. Taken together, these data highlight the broad impact of iAs exposure on the human metabolome, and demonstrate some specificity of the metabolomic response between diabetic and nondiabetic individuals. These data may provide novel insights into the mechanisms and phenotype of diabetes associated with iAs exposure.

The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NAD+, which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg.

Risk assessment of human exposure to chemicals is crucial for understanding whether such agents can cause cancer. The current emphasis on avoidance of animal testing has placed greater importance on in vitro tests for the identification of genotoxicants. Selection of an appropriate in vitro dosing regime is imperative in determining the genotoxic effects of test chemicals. Here, the issue of dosing approaches was addressed by comparing acute and chronic dosing, uniquely using low-dose experiments. Acute 24 h exposures were compared with equivalent dosing every 24 h over 5-day, fractionated treatment periods. The in vitro micronucleus assay was used to measure clastogenicity induced by methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU) in human lymphoblastoid cell line, TK6. Quantitative real-time (qRT) PCR was used to measure mRNA level induction of DNA repair enzymes. Lowest observed genotoxic effect levels (LOGELs) for MMS were obtained at 0.7 µg/ml for the acute study and 1.0 µg/ml for the chronic study. For acute MNU dosing, a LOGEL was observed at 0.46 µg/ml, yet genotoxicity was completely removed following the chronic study. Interestingly, acute MNU dosing demonstrated a statistically significant decrease at 0.009 µg/ml. Levels of selected DNA repair enzymes did not change significantly following doses tested. However, p53 deficiency (using the TK6-isogenic cell line, NH32) increased sensitivity to MMS during chronic dosing, causing this LOGEL to equate to the acute treatment LOGEL. In the context of the present data for 2 alkylating agents, chronic dosing could be a valuable in vitro supplement to acute dosing and could contribute to reduction of unnecessary in vivo follow-up tests.

The growing use of silver nanoparticles (AgNPs) in consumer products raises concerns about potential health effects. This study investigated the persistence and clearance of 2 different size AgNPs (20 and 110 nm) delivered to rats by single nose-only aerosol exposures (6 h) of 7.2 and 5.4 mg/m3, respectively. Rat lung tissue was assessed for silver accumulations using inductively-coupled plasma mass spectrometry (ICP-MS), autometallography, and enhanced dark field microscopy. Involvement of tissue macrophages was assessed by scoring of silver staining in bronchoalveolar lavage fluid (BALF). Silver was abundant in most macrophages at 1 day post-exposure. The group exposed to 20 nm AgNP had the greatest number of silver positive BALF macrophages at 56 days post-exposure. While there was a significant decrease in the amount of silver in lung tissue at 56 days post-exposure compared with 1 day following exposure, at least 33% of the initial delivered dose was still present for both AgNPs. Regardless of particle size, silver was predominantly localized within the terminal bronchial/alveolar duct junction region of the lung associated with extracellular matrix and within epithelial cells. Inhalation of both 20 and 110 nm AgNPs resulted in a persistence of silver in the lung at 56 days post-exposure and local deposition as well as accumulation of silver at the terminal bronchiole alveolar duct junction. Further the smaller particles, 20 nm AgNP, produced a greater silver burden in BALF macrophages as well as greater persistence of silver positive macrophages at later timepoints (21 and 56 days).

Endoplasmic reticulum (ER) stress is recognized as a common theme in the development of metabolic syndrome and other diseases. Chronic liver diseases develop ER stress and also show decreased capacity of drug metabolism. The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. This study was performed to determine whether ER stress condition decreases the expression of PXR and whether the decrease alters the induction of cytochrome P450 3A4 (CYP3A4). Human primary hepatocytes and HepG2 cell line (human hepatocellular carcinoma) were treated with brefeldin A and thapsigargin, 2 well-established ER stressors. Without exceptions, both stressors significantly decreased the expression of PXR. The decrease led to reduced induction of CYP3A4. Reporter dissection study, electrophoretic mobility shift assay, and chromatin immunoprecipitation located in the PXR promoter region 2 adjacent elements recognized by hepatocyte nuclear factor-4α (HNF-4α) and cytidine-cytidine-adenosine-adenosine-thymidine enhanced binding proteins (C/EBPs), respectively. Additional studies demonstrated that HNF-4α was down-regulated during ER stress but the expression of C/EBPβ varied depending on a particular form of C/EBPβ. Liver-enriched activator protein (LAP) was down-regulated but liver-enriched inhibitory protein (LIP) was highly induced. Nevertheless, over-expression of HNF-4α or LAP restored the expression of PXR. Interestingly, the very same sequence also responded to interleukin-6 (IL-6), and primary hepatocytes treated with thapsigargin significantly increased the level of IL-6 mRNA. These findings establish a functional interconnection between ER stress and signaling of proinflammatory cytokines in the regulation of PXR expression.

4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(–/–) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence.

H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (H2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the H2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in H2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate H2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of H2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.

Urinary biomarkers of exposure are used widely in biomonitoring studies. The commonly used urinary biomarkers for the aromatic solvents toluene (T), ethylbenzene (E), and m-xylene (X) are o-cresol, mandelic acid, and m-methylhippuric acid. The toxicokinetics of these biomarkers following inhalation exposure have yet to be described by physiologically based pharmacokinetic (PBPK) modeling. Five male volunteers were exposed for 6 h in an inhalation chamber to 1/8 or 1/4 of the time-weighted average exposure value (TWAEV) for each solvent: toluene, ethylbenzene, and m-xylene were quantified in blood and exhaled air and their corresponding urine biomarkers were measured in urine. Published PBPK model for parent compounds was used and simulations were compared with experimental blood and exhaled air concentration data. If discrepancies existed, Vmax and Km were optimized. Urinary excretion was modeled using parameters found in literature assuming simply stoichiometric yields from parent compound metabolism and first-order urinary excretion rate. Alternative models were also tested for (1) the possibility that CYP1A2 is the only enzyme implicated in o-cresol and (2) a 2-step model for describing serial metabolic steps for mandelic acid. Models adapted in this study for urinary excretion will be further used to interpret urinary biomarker kinetic data from mixed exposures of these solvents.

The Sestrin2 (Sesn2) is an evolutionary conserved enzyme that scavenges reactive oxygen species and regulates autophagy through the AMPK-mTOR pathway. The present study was aimed at determining whether Toll-like receptor (TLR) signaling regulates Sesn2 expression and identifying the underlying molecular mechanism. Lipopolysaccharide (LPS), a representative TLR4 ligand, significantly increased the levels of Sesn2 protein in macrophages. LPS also increased Sesn2 mRNA levels and luciferase reporter activity; however, the mRNA levels of Sesn1 were not affected by LPS. Moreover, treatment of macrophages with other TLR ligands (eg, polyI:C or peptidoglycan) also induced Sesn2 expression. We found that LPS-mediated Sesn2 induction was transcriptionally regulated by AP-1 and Nrf2, and that overexpression of c-Jun or Nrf2 increased Sesn2 protein levels and Sesn2 promoter-driven luciferase reporter activity. Moreover, deletion of the antioxidant response element (ARE) in the Sesn2 promoter or Nrf2 knockout abolished LPS-mediated induction of Sesn2. LPS induced Sesn2 gene expression through p38 and PI3K activation. Surprisingly, treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, caused Sesn2 to accumulate in the cells. In the presence of MG132, we observed that Sesn2 was ubiquitinated. However, LPS treatment attenuated Sesn2 ubiquitination induced by MG132, which resulted in Sesn2 accumulation. Mice treated with d-galactosamine (Gal)/LPS exhibited enhanced Sesn2 expression in the liver. Moreover, infection with a recombinant adenovirus encoding Sens2 markedly reduced the number of Gal/LPS-induced TUNEL-positive cells. Our results suggest that TLR-mediated Sesn2 induction is dependent on AP-1, Nrf2, and the inhibition of ubiquitin-mediated degradation of Sesn2 and might protect cells against endotoxin toxicity.