I started composing these highlights while attending EUROTOX 2104 in Edinburgh, Scotland. Attending this meeting reminded me that Toxicological Sciences, while based in the U.S., is here to serve the entire toxicology community. By way of example, this issue contains papers from the U.S., Spain, Germany, China, Egypt, Portugal, Japan, and the U.K. Mechanistic pathways are generally not up for debate, but how we use the data in these pages to regulate products and processes does involve interpretation and is influenced by the political and cultural landscape. Examining environmental, toxicological, and regulatory challenges from the lenses of different cultures helps avoid provincial thinking. We welcome submissions from our colleagues around the globe. The field benefits from such a global perspective and the collective wisdom that it brings. I invite you to Look Inside ToxSci for the best original research in the field of toxicology. —Gary W. Miller
Aim: Paraquat, a quaternary nitrogen herbicide, is a highly toxic prooxidant resulting in multi-organ failure including the heart although the underlying mechanism still remains elusive. This study was designed to examine the role of the cellular fuel sensor AMP-activated protein kinase (AMPK) in paraquat-induced cardiac contractile and mitochondrial injury. Results: Wild-type and transgenic mice with overexpression of a mutant AMPK α2 subunit (kinase dead, KD), with reduced activity in both α1 and α2 subunits, were administered with paraquat (45 mg/kg) for 48 h. Paraquat elicited cardiac mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic diameter and reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca2+ handling, reduced cell survival, and overt mitochondrial damage (loss in mitochondrial membrane potential). In addition, paraquat treatment promoted phosphorylation of AMPK and autophagy. Interestingly, deficiency in AMPK attenuated paraquat-induced cardiac contractile and intracellular Ca2+ derangement. The beneficial effect of AMPK inhibition was associated with inhibition of the AMPK-TSC-mTOR-ULK1 signaling cascade. In vitro study revealed that inhibitors for AMPK and autophagy attenuated paraquat-induced cardiomyocyte contractile dysfunction. Conclusion: Taken together, our findings revealed that AMPK may mediate paraquat-induced myocardial anomalies possibly by regulating the AMPK/mTOR-dependent autophagy.
The intensive uses of titanium dioxide (TiO2) nanoparticles in sunscreens, toothpaste, sweats, medications, etc. making humans exposed to it daily by not little amounts and also increased its risks including genotoxicity. Thus, the present study was designed as one way to reduce nano-titanium-induced clastogenicity, genotoxicity, and mutagenicity in mice by co-administration of the free radical scavenger chlorophyllin (CHL). In addition, markers of oxidative stress were detected to shed more light on mechanism(s) underlying nano-sized TiO2 genotoxicity. Male mice were exposed to multiple injection into the abdominal cavity for five consecutive days with either CHL (40 mg/kg bw/day), or each of three dose levels of nano-sized TiO2 (500, 1000, or 2000 mg/kg bw/day) alone, or both simultaneously and sacrificed by cervical dislocation 24 h after the last treatment. After CHL co-administration, the observed dose-dependent genotoxicity of TiO2 nanoparticles indicated by the significant elevations in frequencies of both micronuclei and DNA damage induction was significantly decreased and returned to the negative control level. The observed induced mutations in p53 exons 5, 7, & 8 and 5 & 8 in the liver and brain, respectively, were declined in most cases. Moreover, CHL significantly decreased hepatic malondialdehyde level and significantly increased glutathione level and superoxide dismutase, catalase, and glutathione peroxidase activities that were significantly disrupted in animal groups treated with nano-TiO2 alone. In conclusion, the evidenced in vivo genotoxicity of nano-TiO2 in the present study was normalized after CHL co-administration which supports the previously suggested oxidative stress as the possible mechanism for titanium toxicity.
The use of usnic acid and usnic acid-containing products is associated with acute liver failure; however, mechanistic studies of hepatotoxicity caused by usnic acid are limited. In this study, we investigated and characterized the possible mechanisms, especially the role of autophagy in usnic acid's toxicity in human HepG2 cells. Usnic acid caused apoptosis as demonstrated by an increased caspase-3/7 activity and an increased subdiploid nucleus formation. Usnic acid-induced autophagy as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, and an increased number of puncta. Inhibition of autophagy by treating cells with autophagy inhibitors (3-methyladenine or chloroquine) or by small interfering RNA against Atg7 aggravated usnic acid-induced apoptosis and decreased cell viability, indicating that autophagy plays a protective role against usnic acid-induced toxicity. Moreover, usnic acid activated the MAPK signaling pathway. Usnic acid-elicited apoptosis was enhanced and autophagy was decreased when JNK was suppressed by a specific inhibitor. Additionally, inhibition of autophagy decreased the activity of JNK. Taken together, our results suggest that usnic acid perturbs various interrelated signaling pathways and that autophagy induction is a defensive mechanism against usnic acid-induced cytotoxicity.
Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1–4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40–100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.
As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.
The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.
Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2–/– (FA/BRCA-deficient) and FANCD2+/+ (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2+/+ cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMAIII) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2–/– cells. Our results show that preexposure to the trivalent arsenicals MMAIII and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability—measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway.
Adverse drug reactions (ADRs) represent a significant clinical challenge with respect to patient morbidity and mortality. We investigated the hepatotoxicity and systems level metabolic phenotype of methotrexate (MTX) in the context of a prevalent liver disease; non-alcoholic steatohepatitis (NASH). A nuclear magnetic resonance spectroscopic-based metabonomic approach was employed to analyze the metabolic consequences of MTX (0, 10, 40, and 100 mg/kg) in the urine and liver of healthy rats (control diet) and in a model of NASH (methionine-choline deficient diet). Histopathological analysis confirmed baseline (0 mg/kg) liver necrosis, liver inflammation, and lipid accumulation in the NASH model. Administration of MTX (40 and 100 mg/kg) led to liver necrosis in the control cohort, whereas the NASH cohort also displayed biliary hyperplasia and liver fibrosis (100 mg/kg), providing evidence of the synergistic effect of MTX and NASH. The complementary hepatic and urinary metabolic phenotypes of the NASH model, at baseline, revealed perturbation of multiple metabolites associated with oxidative and energetic stress, and folate homeostasis. Administration of MTX in both diet cohorts showed dose-dependent metabolic consequences affecting gut microbial, energy, nucleobase, nucleoside, and folate metabolism. Furthermore, a unique panel of metabolic changes reflective of the synergistic effect of MTX and NASH was identified, including the elevation of hepatic phenylalanine, urocanate, acetate, and both urinary and hepatic formiminoglutamic acid. This systems level metabonomic analysis of the hepatotoxicity of MTX in the context of NASH provided novel mechanistic insight of potential wider clinical relevance for further understanding the role of liver pathology as a risk factor for ADRs.
The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI).
The target site for inhaled vapor-induced injury often differs in mouth-breathing humans compared with nose-breathing rats, thus complicating the use of rat inhalation toxicity data for assessment of human risk. We sought to examine sensitivity of respiratory/transitional nasal (RTM) and tracheobronchial (TBM) mucosa to two electrophilic irritant vapors: diacetyl and acrolein. Computational fluid dynamic physiologically based pharmacokinetic modeling was coupled with biomarker assessment to establish delivered dose–response relationships in RTM and TBM in male F344 rats following 6 h exposure to diacetyl or acrolein. Biomarkers included glutathione status, proinflammatory and antioxidant gene mRNA levels, and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Modeling revealed that 0.0094–0.1653 μg acrolein/min-cm2 and 3.9–21.6 μg diacetyl/min-cm2 were deposited into RTM/TBM. Results indicate RTM and TBM were generally of similar sensitivity to diacetyl and acrolein. For instance, both tissues displayed induction of antioxidant and proinflammatory genes, and nuclear accumulation of Nrf2 after electrophile exposure. Hierarchical cellular response patterns were similar in RTM and TBM but differed between vapors. Specifically, diacetyl exposure induced proinflammatory and antioxidant genes concomitantly at low exposure levels, whereas acrolein induced antioxidant genes at much lower exposure levels than that required to induce proinflammatory genes. Generally, diacetyl was less potent than acrolein, as measured by maximal induction of transcripts. In conclusion, the upper and lower extrapulmonary airways are of similar sensitivity to inhaled electrophilic vapors. Dosimetrically based extrapolation of nasal responses in nose-breathing rodents may provide an approach to predict risk to the lower airways of humans during mouth-breathing.
High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000–1500-ppm NaPB resulted in plasma levels around 3–5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.
Methylmercury, polychlorinated biphenyls (PCBs), and perfluorinated compounds (PFCs) are ubiquitous and persistent environmental chemicals with known or suspected toxic effects on the nervous system and the immune system. Animal studies have shown that tissue damage can elicit production of autoantibodies. However, it is not known if autoantibodies similarly will be generated and detectable in humans following toxicant exposures. Therefore, we conducted a pilot study to investigate if autoantibodies specific for neural and non-neural antigens could be detected in children at age 7 years who have been exposed to environmental chemicals. Both prenatal and age-7 exposures to mercury, PCBs, and PFCs were measured in 38 children in the Faroe Islands who were exposed to widely different levels of these chemicals due to their seafood-based diet. Concentrations of IgM and IgG autoantibodies specific to both neural (neurofilaments, cholineacetyltransferase, astrocyte glial fibrillary acidic protein, and myelin basic protein) and non-neural (actin, desmin, and keratin) antigens were measured and the associations of these autoantibody concentrations with chemical exposures were assessed using linear regression. Age-7 blood-mercury concentrations were positively associated with titers of multiple neural- and non-neural-specific antibodies, mostly of the IgM isotype. Additionally, prenatal blood-mercury and -PCBs were negatively associated with anti-keratin IgG and prenatal PFOS was negatively associated with anti-actin IgG. These exploratory findings demonstrate that autoantibodies can be detected in the peripheral blood following exposure to environmental chemicals. The unexpected association of exposures with antibodies specific for non-neural antigens suggests that these chemicals may have toxicities that have not yet been recognized.
The mycotoxin deoxynivalenol (DON) elicits robust anorectic and emetic effects in several animal species. However, less is known about the potential for naturally occurring and synthetic congeners of this trichothecene to cause analogous responses. Here we tested the hypothesis that alterations in DON structure found in the plant metabolite deoxynivalenol-3-glucoside (D3G) and two pharmacologically active synthetic DON derivatives, EN139528 and EN139544, differentially impact their potential to evoke food refusal and emesis. In a nocturnal mouse food consumption model, oral administration with DON, D3G, EN139528, or EN139544 at doses from 2.5 to 10 mg/kg BW induced anorectic responses that lasted up to 16, 6, 6, and 3 h, respectively. Anorectic potency rank orders were EN139544>DON>EN139528>D3G from 0 to 0.5 h but DON>D3G>EN139528>EN139544 from 0 to 3 h. Oral exposure to each of the four compounds at a common dose (2.5 mg/kg BW) stimulated plasma elevations of the gut satiety peptides cholecystokinin and to a lesser extent, peptide YY3–36 that corresponded to reduced food consumption. In a mink emesis model, oral administration of increasing doses of the congeners differentially induced emesis, causing marked decreases in latency to emesis with corresponding increases in both the duration and number of emetic events. The minimum emetic doses for DON, EN139528, D3G, and EN139544 were 0.05, 0.5, 2, and 5 mg/kg BW, respectively. Taken together, the results suggest that although all three DON congeners elicited anorectic responses that mimicked DON over a narrow dose range, they were markedly less potent than the parent mycotoxin at inducing emesis.
Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity.
Methotrexate (MTX) is used for rheumatoid arthritis (RA) treatment showing a wide toxicity profile. This study aimed to evaluate the influence of single nucleotide polymorphisms (SNPs) in genes encoding for MTX transporters with the occurrence of MTX-related toxicity (overall and gastrointestinal). A total of 233 Portuguese RA patients were genotyped for 23 SNPs. Haplotype analyses were performed and a toxicogenetic risk index (TRI) was created for SNPs that revealed to be statistically significant. Regarding MTX overall toxicity, an increased risk was associated to SLC19A1 rs7499 G carriers (p = 0.017), SLC46A1 rs2239907 GG (p = 0.030) and, SLCO1B1 rs4149056 T carriers (p = 0.040) and TT (p = 0.019). TRI revealed that patients with Index 3 were 18-fold more likely to present an adverse drug reaction when compared to those with Index 1 (p = 0.001). For MTX gastrointestinal toxicity, results demonstrated an increased risk associated with SLC19A1 rs7499 G carriers (p = 0.012) and GG (p = 0.045), SLC19A1 rs1051266 G carriers (p = 0.034), SLC19A1 rs2838956 A carriers (p = 0.049) and, SLCO1B1 rs4149056 T carriers (p = 0.042) and TT (p = 0.025). Haplotype analyses showed association between GGAG haplotype for SLC19A1 rs7499, rs1051266, rs2838956 and rs3788200 with MTX gastrointestinal toxicity (p = 0.029). TRI revealed that patients with Index 4 were 9-fold more likely to present a gastrointestinal disorder when compared to those with Index 1 (p = 0.020). This study demonstrated that SLC19A1, SLC46A1 and SLCO1B1 genotypes may help to identify patients with increased risk of MTX-related overall toxicity and that SLC19A1 and SLCO1B1 genotypes, and SLC19A1 haplotypes may help to identify patients with increased risk of MTX-related gastrointestinal toxicity.
Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework.
Arsenic exposure during embryonic development can cause ischemic heart pathologies later in adulthood which may originate from impairment in proper blood vessel formation. The arsenic-associated detrimental effects are mediated by arsenite (iAsIII) and its most toxic metabolite, monomethylarsonous acid [MMA (III)]. The impact of MMA (III) on coronary artery development has not yet been studied. The key cellular process that regulates coronary vessel development is the epithelial-mesenchymal transition (EMT). During cardiac EMT, activated epicardial progenitor cells transform to mesenchymal cells to form the cellular components of coronary vessels. Smad2/3 mediated TGFβ2 signaling, the key regulator of cardiac EMT, is disrupted by arsenite exposure. In this study, we compared the cardiac toxicity of MMA (III) with arsenite. Epicardial progenitor cells are 15 times more sensitive to MMA (III) cytotoxicity when compared with arsenite. MMA (III) caused a significant blockage in epicardial cellular transformation and invasion at doses 10 times lower than arsenite. Key EMT genes including TGFβ ligands, TβRIII, Has2, CD44, Snail1, TBX18, and MMP2 were down regulated by MMA (III) exposure. MMA (III) disrupted Smad2/3 activation at a dose 20 times lower than arsenite. Both arsenite and MMA (III) significantly inhibited Erk1/2 and Erk5 phosphorylation. Nuclear translocation of Smad2/3 and Erk5 was also blocked by arsenical exposure. However, p38 activation, as well as smooth muscle differentiation, was refractory to the inhibition by the arsenicals. Collectively, these findings revealed that MMA (III) is a selective disruptor of cardiac EMT and as such may predispose to arsenic-associated cardiovascular disorders.
The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MSE approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MSE approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.
The role of the multi-resistance protein 2 (Mrp2) in the nephropathy induced by inorganic mercuric mercury (Hg2+) was studied in rats (TR–) and mice (Mrp2–/–), which lack functional Mrp2, and control animals. Animals were exposed to nephrotoxic doses of HgCl2. Forty-eight or 24 hours after exposure, tissues were harvested and analyzed for Hg content and markers of injury. Histological analyses revealed that the proximal tubular segments affected pathologically by Hg2+ were significantly different between Mrp2-deficient animals and controls. In the absence of Mrp2, cellular injury localized almost exclusively in proximal tubular segments in the subcapsular (S1) to midcortical regions (early S2) of the kidney. In control animals, cellular death occurred mainly in the proximal tubular segments in the inner cortex (late S2) and outer stripe of the outer medulla (S3). These differences in renal pathology indicate that axial heterogeneity exists along the proximal tubule with respect to how mercuric ions are handled. Total renal and hepatic accumulation of mercury was also greater in animals lacking Mrp2 than in controls, indicating that Mrp2 normally plays a significant role in eliminating mercuric ions from within proximal tubular cells and hepatocytes. Analyses of plasma creatinine, BUN, and renal expression of Kim-1 and Ngal tend to support the severity of the nephropathies detected histologically. Collectively, our findings indicate that a fraction of mercuric ions is normally secreted by Mrp2 in early portions of proximal tubules into the lumen and then is absorbed downstream in straight portions, where mercuric species typically induce toxic effects.
Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.
Occupational and environmental exposure to tri-cresyl phosphates (TCPs) may cause various types of neurotoxicity. Among the TCP isomers, tri-ortho-cresyl phosphate is a well-studied organophosphate (OP) known to cause OP-induced delayed neuropathy (OPIDN). Clinically, OPIDN is characterized by limb paralysis caused by the inhibition of neuropathy target esterase. Like other OPs, TOCP may also trigger acute toxicity by yet unknown mechanisms. Neurotoxic effects of TCPs, including TOCP, on central nervous system functions have not been studied in depth, and such non-OPIDN mechanisms might be related to the aerotoxic syndrome. To identify alternative mechanisms of TOCP neurotoxicity, we conducted an in vitro study using primary cortical neurons isolated from mouse embryos (E 16.5). After 24 h or 6 days in vitro (DIV), cell cultures were treated with different TOCP concentrations for 24 h. On DIV 2 and 7, we investigated three different endpoints—general cytotoxicity, neurite outgrowth, and glutamatergic signaling. At both time points, the EC50 for TOCP-induced cell death was 90 μM, however, neurite outgrowth was already significantly affected at TOCP concentrations of 10 μM. The number of cells responding to glutamate, as well as the corresponding mean response amplitudes were reduced with TOCP concentrations as low as 100 nM. For the first time, functional neurotoxicity is observed with very low TOCP concentrations, and in the absence of structural damages. Our proposed mechanism is that TOCP exposure may lead to cognitive deficits relevant in aerotoxic syndrome by inhibiting the signaling of glutamate, the most abundant excitatory neurotransmitter in the brain.
Amiodarone (AD) is a highly efficient antiarrhythmic drug with potentially serious side effects. Severe pulmonary toxicity is reported in patients receiving AD even at low doses and may cause interstitial pneumonia as well as lung fibrosis. Apoptosis of alveolar epithelial type II cells (AECII) has been suggested to play an important role in this disease. In the current study, we aimed to establish a murine model of AD-induced lung fibrosis and analyze surfactant homeostasis, lysosomal, and endoplasmic reticulum (ER) stress in this model. AD/vehicle was instilled intratracheally into C57BL/6 mice, which were sacrificed on days 7, 14, 21, and 28. Extent of lung fibrosis development was assessed by trichrome staining and hydroxyproline measurement. Cytotoxicity was assessed by lactate dehydrogenase assay. Phospholipids (PLs) were analyzed by mass spectrometry. Surfactant proteins (SP) and markers for apoptosis, lysosomal, and ER stress were studied by Western blotting and immunohistochemistry. AECII morphology was evaluated by electron microscopy. Extensive lung fibrosis and AECII hyperplasia were observed in AD-treated mice already at day 7. Surfactant PL and SP accumulated in AECII over time. In parallel, induction of apoptosis, lysosomal, and ER stress was encountered in AECII of mice lungs and in MLE12 cells treated with AD. In vitro, siRNA-mediated knockdown of cathepsin D did not alter the AD-induced apoptotic response. Our data suggest that mice exposed to intratracheal AD develop severe pulmonary fibrosis, exhibit extensive surfactant alterations and cellular stress, but AD-induced AECII apoptosis is not mediated primarily via cathepsin D.
The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1 in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1 was shown in vitro and in a bile-duct cannulated rat 14C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3, was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1 to rats significantly reduced the formation of M3 and prevented liver toxicity, whereas direct administration of M3 reproduced the toxicity. Structural changes were introduced to 1 to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2), and addition of a methyl group to the benzylic carbon (compound 3) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3 (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.