Toxicological Sciences - Advance Access

Roles of the Genetic Polymorphisms of Alcohol-metabolizing Enzymes on the Immunology in High-risk Drinkers

2009-06-30

Alcohol metabolism involves several enzymes and the individual genetic variations in the alcohol metabolism are related to the absorption, distribution and elimination of alcohol and metabolites such as acetaldehyde. Therefore, the genetic variations of alcohol-metabolizing enzymes are responsible for the different toxicity of alcohol in several organs like liver and immunological systems. The purpose of this study was to evaluate if the life styles such as drinking and smoking and the genetic variations of alcohol-metabolizing enzymes (ADH2, ALDH2, CYP2E1, and CAT) were associated with the immunological biomarkers. In this study, one hundred and five high-risk drinkers and 102 low-risk drinkers who were excluded from the immuno-related diseases and other critical diseases were enrolled to evaluate the immunological functions. Counts of white blood cells, mononuclear cells, and lymphocyte subpopulations, and liver and immunological function tests were measured. Genotypes of alcohol-metabolizing enzymes were assayed by a real-time PCR and PCR-RFLP. We found that there were significantly higher percentages of CD4⁺ T lymphocytes, ratios of CD4⁺/CD8⁺ and AST/ALT, activities of AST and GGT, and levels of IL-6, but lower percentages of CD8⁺ T lymphocytes, levels of total protein, globulin, and immunoglobulins in the high-risk drinkers. In addition, the synergistic effects of smoking and drinking on the count of WBC and mononuclear cells were found to be significant. Furthermore, there were higher OR to become high-risk drinkers in subjects with the combination of ALDH2 (*1/*1) genotype and either genotype of ADH2 or CYP2E1 than the others with other combinations of genotypes. Additionally, there were more abnormal immunological tests in the subjects with higher activity of ADH2 and lower activity of ALDH2. Our results suggested that the habits of drinking, smoking, and betel chewing, and genetic variations of alcohol metabolism were associated with the immunological biomarkers.

Cellular Stress Response Pathway System as a Sentinel Ensemble in Toxicological Screening

Simmons, S. O., Fan, C.-Y., Ramabhadran, R. — 2009-06-30

High costs, long test times and societal concerns related to animal use have required the development of in vitro assays for the rapid and cost-effective toxicological evaluation and characterization of compounds in both the pharmaceutical and environmental arenas. While the pharmaceutical industry has developed very effective, high-throughput in vitro assays for determining the therapeutic potential of compounds, the application of this approach to toxicological screening has been limited. A primary reason for this is that while drug efficacy searches are directed to a target/mechanism, xenobiotics can cause toxicity through any of a myriad of undefined interactions with cellular components and processes. Given that it is not practical to design assays that can interrogate each potential toxicological target, an integrative approach is required if there is to be a rapid and low-cost toxicological evaluation of chemicals. Cellular stress response pathways offer a viable solution to the creation of a set of integrative assays as there is a limited and hence manageable set (a small ensemble of 10 or less) of major cellular stress response pathways through which cells mount a homoeostatic response to toxicants and which also participate in cell fate/death decisions. Further, over the past decades, these pathways have been well characterized at a molecular level thereby enabling the development of high throughput cell-based assays using the components of the pathways. Utilization of the set of cellular stress response pathway-based assays as indicators of toxic interactions of chemicals with basic cellular machinery will potentially permit the clustering of chemicals based on biological response profiles of common mode of action (MOA) and also the inference of the specific MOA of a toxicant.

This article reviews the biochemical characteristics of the stress response pathways, their common architecture that enables rapid activation during stress, their participation in cell fate decisions, the essential nature of these pathways to the organism, and the biochemical basis of their cross-talk that permits an assay ensemble screening approach. Subsequent sections describe how the stress pathway ensemble assay approach could be applied to screening potentially toxic compounds and discuss how this approach may be used to derive toxicant MOA from the biological activity profiles that the ensemble strategy provides. The article concludes with a review of the application of the stress assay concept to non-invasive in vivo assessments of chemical toxicants.

Domoic acid induces a long-lasting enhancement of CA1 field responses and impairs tetanus-induced long-term potentiation in rat hippocampal slices

Qiu, S., Jebelli, A. K., Ashe, J. H., Curras-Collazo, M. C. — 2009-06-29

Domoic acid (DOM) is known to cause hippocampal neuronal damage and produces amnesic effects. We examined synaptic plasticity changes induced by DOM exposure in rat hippocampal CA1 region. Brief bath application of DOM to hippocampal slices produces a chemical form of long-term potentiation (LTP) of CA1 field synaptic potentials. The potentiation cannot be blocked by NMDA receptor antagonist MK-801, but can be blocked by the calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-62 or cAMP-dependent protein kinase (PKA) inhibitor H-89. DOM-potentiated slices show decreased autophosphorylated CaMKII (p-Thr286), an effect that is also dependent on the activity of CaMKII and PKA. Increased phosphorylation of AMPA receptor subunit GluR1 (p-Ser831) was seen in DOM potentiated slices. Therefore, aberrant regulation of CaMKII and GluR1 phosphorylation occurs after DOM application. In addition, tetanus-induced LTP as well as the increase of phosphorylation of CaMKII (p-Thr286) was reduced in DOM-potentiated slices. Compared with brief exposure, slices recovering from prolonged exposure did not show potentiation or altered levels of CaMKII (p-Thr286) or GluR (p-Ser831). However, decreased phosphorylation of GluR1 at Ser845 was seen. These results describe a new chemical form of LTP and uncover novel molecular changes induced by DOM. The observed impairment of tetanus LTP and misregulation of CaMKII and GluR1 phosphorylation may partially account for DOM neurotoxicity and underlie the molecular basis for DOM-induced memory deficit.

Cumulative and antagonistic effects of a mixture of the antiandrogens vinclozolin and iprodione in the pubertal male rat

2009-06-29

Vinclozolin and iprodione are dicarboximide fungicides that display anti-androgenic effects in the male rat, which suggests a mixture would lead to cumulative effects on androgen-sensitive endpoints. Iprodione is a steroid synthesis inhibitor, but AR antagonist activity, which is displayed by vinclozolin, has not been fully evaluated. Here, we demonstrate that iprodione binds to the hAR (IC₅₀ = 86.0 µM), reduces androgen-dependent gene expression, and reduces androgen sensitive tissue weights in castrated male rats (Hershberger assay). Since vinclozolin and iprodione affect common targets in the pubertal male rat, we tested the hypothesis that a mixture would have cumulative anti-androgenic effects. An iprodione dose, that does not significantly affect androgen-dependent morphological endpoints, was combined with vinclozolin doses (2x5 factorial design). Sprague-Dawley rats were dosed by gavage with vinclozolin at 0, 10, 30, 60, and 100 mg/kg/d with and without 50 mg iprodione/kg/d from postnatal day (PND) 23 to 55-57 (n = 8/group). The age at puberty (preputial separation (PPS)), organ weights, serum hormones, and ex vivo testis steroid hormone production were measured. Vinclozolin delayed PPS, reduced androgen sensitive organ weights, and increased serum testosterone. The addition of iprodione enhanced the vinclozolin inhibition of PPS (PND 47.5 vs 49.1; 2-way ANOVA: iprodione main effect p = 0.0002). The dose response for several reproductive and non-reproductive organ weights was affected in a cumulative manner. In contrast, iprodione antagonized the vinclozolin-induced increase in serum testosterone. These results demonstrate these fungicides interact on common targets in a tissue-specific manner when co-administered to the pubertal male rat.

Does Exposure to Perfluoroalkyl Acids Present a Risk to Human Health?

Rosen, M. B., Lau, C., Corton, J. C. — 2009-06-26

Effects of Nitric Oxide and Antioxidants on Advanced Glycation End Products-Induced Hypertrophic Growth in Human Renal Tubular Cells

Huang, J.-S., Chuang, L.-Y., Guh, J.-Y., Huang, Y.-J. — 2009-06-24

The accumulation of advanced glycation end products (AGE) is a key mediator of renal tubular hypertrophy in diabetic nephropathy (DN). Reactive oxygen species (ROS) and nitric oxide (NO) were involved in the progression of DN. In this study, the molecular mechanisms of NO and antioxidants responsible for inhibition of AGE-induced renal tubular hypertrophy were examined. We found that AGE (but not non-glycated bovine serum albumin) significantly suppressed the NO/cGMP/PKG signaling in human renal proximal tubular cells. NO donors S-nitroso-N-acetylpenicillamine (SNAP)/sodium nitroprusside (SNP) and antioxidants N-acetylcysteine (NAC)/taurine treatments significantly attenuated AGE-inhibited NO production, cGMP synthesis, and inducible NO synthase (iNOS)/cGMP-dependent protein kinase (PKG) activation. Moreover, AGE-induced the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) activation were markedly blocked by anti-receptor for AGE (RAGE), SNAP, SNP, NAC, and taurine. The abilities of NO and antioxidants to inhibit AGE/RAGE-induced hypertrophic growth were verified by the observation that SNAP, SNP, NAC, and taurine inhibited fibronectin, p21^Waf1/Cip1, and RAGE expression. Therefore, antioxidants significantly attenuated AGE/RAGE-enhanced cellular hypertrophy partly through induction of the NO/cGMP/PKG signaling.

Acrylonitrile-induced Oxidative Stress and Oxidative DNA Damage in Male Sprague-Dawley Rats

Pu, X., Kamendulis, L. M., Klaunig, J. E. — 2009-06-22

Studies have demonstrated that the induction of oxidative stress may be involved in brain tumor induction in rats by acrylonitrile. The present study examined whether acrylonitrile induces oxidative stress and DNA damage in rats, and whether blood can serve as a valid surrogate for the biomonitoring of oxidative stress induced by acrylonitrile in the exposed population. Male Sprague-Dawley rats were treated with 0, 3, 30, 100, and 200 ppm acrylonitrile in drinking water for 28 days. One group of rats were also coadministered N-acetyl cysteine (0.3% in diet) with acrylonitrile (200ppm in drinking water) to examine whether antioxidant supplementation was protective against acrylonitrile-induced oxidative stress. Direct DNA strand breakage in white blood cells (WBC) and brain was measured using the alkaline Comet assay. Oxidative DNA damage in WBC and brain was evaluated using formamidopyrimidine (fpg)-modified Comet assay and with HPLC-electrochemical detection. No significant increase in direct DNA strand breaks was observed in brain and WBC from acrylonitrile treated rats. However, oxidative DNA damage (fpg-Comet and OH8dG) in brain and WBC was increased in a dose-dependent manner. In addition, plasma levels of reactive oxygen species (ROS) increased in rats administered acrylonitrile. Dietary supplementation with N-acetyl cysteine prevented acrylonitrile-induced oxidative DNA damage in brain and WBC. A slight, but significant, decrease in the GSH/GSSG ratio was seen in brain at acrylonitrile doses > 30 ppm. These results provide additional support that the mode of action for acrylonitrile-induced astrocytomas involves the induction of oxidative stress and damage. Significant associations were seen between oxidative DNA damage in white blood cells and brain, ROS formation in plasma and the reported tumor incidences. Since oxidative DNA damage in brain correlated with oxidative damage in WBC, these results suggest that monitoring WBC DNA damage maybe a useful tool to assess acrylonitrile-induced oxidative stress in humans.

Potential Role of {alpha}-Synuclein and Metallothionein in Lead-Induced Inclusion Body Formation

Zuo, P., Qu, W., Cooper, R. N., Goyer, R. A., Diwan, B. A., Waalkes, M. P. — 2009-06-19

Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knock-out (MT-null) and parental wild type (WT) cell lines to further explore the formation process of Pb induced IBs. Unlike WT cells, MT-null cells did not form IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT cells was lost during Pb exposure as IBs formed. Transfection of MT-I into MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced IBs may be like disease-related aggresomes, which often contain -synuclein (Scna), we investigated Scna expression in cells capable (WT) and incapable (MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal expression in MT-null cells. Pb exposure increased Scna expression only in WT cells. MT-transfection increased Scna transcript to WT levels. In WT or MT-transfected MT-null cells, Pb-induced Scna expression rapidly increased and then decreased over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and MT was confirmed ex vivo by antibody pulldown assay where the proteins co-precipitated with an antibody to MT. Pb exposure caused increased co-localization of MT and Scna proteins with time only in WT cells. In WT mice after chronic Pb exposure Scna was localized in renal cells containing forming IBs while MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced IBs and, with MT, may play a role in IBs formation.

Long-term exposure to zidovudine delays cell cycle progression, induces apoptosis, and decreases telomerase activity in human hepatocytes

Fang, J.-L., Beland, F. A. — 2009-06-18

Zidovudine (3’-azido-3’-deoxythymidine; AZT), which is currently used in the treatment of acquired immunodeficiency syndrome, has been shown to have anticancer properties. In the present study, we examined the mechanisms contributing to increased sensitivity of cancer cells to the growth-inhibitory effects of AZT. This was accomplished by incubating a hepatoma cell line (HepG2) and a normal liver cell line (THLE2) with AZT in continuous culture for up to 4 weeks and evaluating the number of viable and necrotic cells, the induction of apoptosis, cell cycle alterations, and telomerase activity. In HepG2 cells, AZT (2 - 100 µM) caused significant dose-dependent decreases in the number of viable cells at exposures > 24 h. During a 1-week recover period, there was only a slight increase in the number of viable cells treated with AZT. The decrease in viable cells was associated with an induction of apoptosis, a decrease in telomerase activity, and S and G2/M phase arrest of the cell cycle. During the recovery period, the extent of apoptosis and telomerase activity returned to control levels, while the disruption of cell cycle progression persisted. Western blot analysis indicated that AZT caused a decrease in checkpoint kinase 1 (Chk1) and kinase 2 (Chk2) and an increase in phosphorylated Chk1 (Ser345) and Chk2 (Thr68). Similar effects, to lesser extent, were observed in THLE2 cells given much higher concentrations of AZT (50 - 2,500 µM). These data show that HepG2 cells are much more sensitive than THLE2 cells to AZT. They also indicate that a combination of a delay of cell cycle progression, an induction of apoptosis, and a decrease in telomerase activity is contributing to the decrease in the number of viable cells from AZT treatment, and that checkpoint enzymes Chk1 and Chk2 may play an important role in the delay of cell cycle progression.

Atrazine Oral Exposure of Peripubertal Male Rats Down-Regulates Steroidogenesis Gene Expression in Leydig Cells

Pogrmic, K., Fa, S., Dakic, V., Kaisarevic, S., Kovacevic, R. — 2009-06-18

In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50mg/kg and 200mg/kg body weight daily from postnatal day 23 to 50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein, translocator protein, steroidogenic factor-1, phosphodiesterase 4B, 3β–hydroxysteroid dehydrogenase (HSD), CYP17A1 and 17βHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extra cellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was down-regulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMP-dependent genes. To clarify the activity of the steroidogenic enzymes responsible for androgenesis, purified Leydig cells were challenged with different steroid substrates (22OH-cholesterol, pregnenolone, progesterone and ⁴–androstenedione), and the obtained results indicated inhibition of androgen production in Leydig cells isolated from atrazine-treated animals in the presence of all those substrates. However, when Leydig cells were challenged with 22OH-cholesterol, the progesterone level in the incubation medium was unchanged, indicating that decrease in cholesterol transport and/or CYP17A1 and 17βHSD activity are most probably responsible for inhibition of androgen production after the addition of different substrates. Our results demonstrated that in vivo exposure to atrazine affects Leydig cell steroidogenesis via the inhibition of steroidogenesis gene expression, which is accompanied by decreased androgenesis.

Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondria involves PTPC-dependent activation of the mitochondrial pathway of apoptosis

2009-06-18

Mycotoxins produced by the Fusarium molds can cause a variety of human diseases and economic losses in livestock. Fusaria produce predominantly two types of mycotoxins: the non-estrogenic trichotecenes including T-2 toxin and the mycoestrogens such as Zearalenone (ZEN). In a previous report, we demonstrated that the hepatotoxicity of these mycotoxins involves the mitochondrial pathway of apoptosis. Here, we observed that both fusarotoxins induced cell death by a mitochondria-dependent apoptotic process which includes opening of the mitochondrial permeability transition pore (PTPC), loss of mitochondrial transmembrane potential (_m), increase in O₂^.– production, mitochondrial relocalisation of Bax, cytochrome c release and caspase activation. Studies performed on isolated mouse liver mitochondria showed that both ZEN and T-2 toxin might act directly on mitochondria to induce a PTPC-dependent permeabilization of mitochondrial membranes. Moreover, they may target different members of PTPC. Indeed, while the inner membrane protein ANT could be the target of T-2 toxin, ZEN seems to target the outer membrane protein VDAC. Cells pre-treatment with the p53 inhibitor pifithrin- (PFT) suggested that ZEN but not T-2 toxin, triggered a p53-dependent mitochondrial apoptotic pathway. Finally, mitochondrial alterations induced by ZEN and T-2 toxin are mediated by Bcl-2 family proteins such as Bax, and prevented by Bcl-X_L and to a lesser extent by Bcl-2. Taken together, these data indicate that mitochondria play a pivotal role in both ZEN and T-2 toxin-induced apoptosis and that PTPC members and proteins of Bcl-2 family should be interesting targets to overcome fusarotoxin toxicity.

Effects of Decabrominated Diphenyl Ether (PBDE 209) Exposure at Different Developmental Periods on Synaptic Plasticity in the Dentate Gyrus of Adult Rats in vivo

Xing, T., Chen, L., Tao, Y., Wang, M., Chen, J., Ruan, D.-Y. — 2009-06-17

Polybromininated diphenyl ethers (PBDEs) are widely used as flame-retardant additives. Previous studies have demonstrated that PBDEs exposure can lead to neurotoxicity. However, little is known about the effects of PBDE 209 on synaptic plasticity. This study investigated the effect of decabrominated diphenyl ether (PBDE 209), a major PBDEs product, on synaptic plasticity in the dentate gyrus (DG) of rats at different developmental periods. We examined the input/output functions (I/O), paired-pulse reactions (PPR) and the long-term potentiation (LTP) of the field excitatory postsynaptic potential (fEPSP) slope and the population spike (PS) amplitude in vivo. Rats were exposed to PBDE 209 during five different developmental periods: pregnancy, lactation via mother's milk, lactation via intragastric administration, after weaning, and prenatal to life. We found that exposed to PBDE 209 during different developmental periods could impair the synaptic plasticity of adult rats in different degrees. The results also showed that PBDE 209 might cause more serious effects on the postsynaptic cell excitability in synaptic plasticity, and the lactation period was the most sensitive time of development towards PBDE 209.

Pubertal administration of DEHP delays puberty, suppresses testosterone production and inhibits reproductive tract development in male Sprague-Dawley and Long-Evans Rats

2009-06-15

While is clear that exposure to high dosage levels of some phthalates delays the onset of puberty in the male rat it has been hypothesized that low levels of DEHP accelerate puberty by enhancing testicular androgen synthesis. The current study was designed to determine if the dose response to DEHP was non-monotonic, as hypothesized. Pubertal administration of DEHP delayed the onset of puberty and reduced androgen-dependent tissue weights in both Long Evans (LE) and Sprague Dawley (SD) male rats 300 and 900 mg DEHP/kg/d. These effects were generally of greater magnitude in LE than SD rats. By contrast, alterations in testis histopathology (300 and 900 mg/kg/d) were more severe in SD than in LE rats. Taken together, these results suggest that DEHP may be acting on the pubertal male rat testis via two modes of action; one via the Leydig cells and the other via the Sertoli cells.

Treatment with DEHP generally reduced serum testosterone and increased serum LH levels, demonstrating that the reduction in testosterone was due to the effect of DEHP on the testis and not via an inhibition of LH from hypothalamic-pituitary axis. Testosterone production ex vivo (with and without hCG stimulation) was consistently reduced in males at the time of puberty and shortly thereafter. DEHP treatment did not accelerate the age at puberty or enhance testosterone levels at 10 or 100 mg/kg/d in either LE or SD rats, as some have hypothesized. Taken together, these results do not provide any evidence of a non-monotonic dose response to DEHP during puberty.

Hemangiosarcoma in Rodents: Mode-of-Action Evaluation and Human Relevance

2009-06-12

Although rarely occurring in humans, hemangiosarcomas (HS) have become important in evaluating the potential human risk of several chemicals, including industrial, agricultural, and pharmaceutical agents. Spontaneous hemangiosarcomas arise frequently in mice, less commonly in rats, and frequently in numerous breeds of dogs. This review explores knowledge gaps and uncertainties related to the mode of action (MOA) for the induction of hemangiosarcomas in rodents, and evaluates the potential relevance for human risk. For genotoxic chemicals (vinyl chloride and thorotrast), significant information is available concerning the MOA. In contrast, numerous chemicals produce hemangiosarcomas in rodents by non-genotoxic, proliferative mechanisms. An overall framework is presented, including direct and indirect actions on endothelial cells, paracrine effects in local tissues, activation of bone marrow endothelial precursor cells, and tissue hypoxia. Numerous obstacles are identified in investigations into the MOA for mouse hemangiosarcomas and the relevance of the mouse tumors to humans, including lack of identifiable precursor lesions, usually late occurrence of the tumors, and complexities of endothelial biology. This review proposes a working MOA for HS induced by non-genotoxic compounds that can guide future research in this area. Importantly, a common MOA appears to exist for the non-genotoxic induction of HS, where there appears to be a convergence of multiple initiating events (e.g., hemolysis, decreased respiration, adipocyte growth) leading to either dysregulated angiogenesis and/or erythropoiesis that produces hypoxia and macrophage activation. These later events lead to the release of angiogenic growth factors and cytokines that stimulate endothelial cell proliferation, which, if sustained, provide the milieu that can lead to HS formation.

Octachlorostyrene Induces Cytochrome P450, UDP-glucuronosyltransferase and Sulfotransferase via the Aryl Hydrocarbon Receptor and Constitutive Androstane Receptor

Yanagiba, Y., Ito, Y., Kamijima, M., Gonzalez, F. J., Nakajima, T. — 2009-06-12

Octachlorostyrene (OCS) is a by-product produced in the process of synthesis of chlorinated compounds. There are some reports concerning environmental contamination by OCS, but few on the toxicological effects on human. Drug-metabolizing enzymes may play an important role in toxicity through metabolic activation or deactivation of OCS. In this study, we investigated whether OCS influences these enzymes using wild-type and aryl hydrocarbon receptor (Ahr)-null mice; AhR regulates cytochrome P450 (CYP) 1A, UDP-glucuronosyltransferase (UGT) or sulfotransferase (SULT). Both mouse lines were treated with OCS (0, 32 and 64 µmol/kg) for 4 days by gavage. As a reference, the mice were treated with 20 mg/kg 3-methylcholanthrene (3MC) for 4 days. OCS treatment increased the expression of CYP 1A1 and CYP1A2 mRNA and ethoxyresorfin O-deethylase activity only in the wild-type mice, similar to that of the AhR activator 3MC. OCS treatment increased expression of UGT1A6 and SULT 1A1 mRNA and their associated enzyme activities only in Ahr-null mice, while 3MC still influenced these enzymes only in wild-type mice. OCS induced constitutive androstane receptor (CAR) only in Ahr-null mice, and the target gene CYP2B10 mRNA was induced more strongly in Ahr-null mice than in wild-type mice. 3MC slightly induced CYP2B10 mRNA only in the wild-type mice. These results suggest that CAR is involved in regulation of the UGT and SULT genes by OCS. Thus, OCS may regulate CYP1A via AhR, while it controls UGT1A6 and SULT1A via CAR.

Inhibition of UV-C light-induced apoptosis in liver cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). -Inhibition of apoptosis by TCDD-

Chopra, M., Dharmarajan, A. M., Meiss, G., Schrenk, D. — 2009-06-10

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic pollutant ubiquitously present in the environment. Most of the toxic effects of TCDD are believed to be mediated by high-affinity binding to the aryl hydrocarbon receptor (AhR) and subsequent effects on gene transcription. TCDD causes cancer in multiple tissues in different animal species and is classified as a class 1 human carcinogen. In initiation-promotion studies TCDD was shown to be a potent liver tumor-promotor. Among other theories it has been hypothesized that TCDD acts as a tumor-promotor by preventing initiated cells from undergoing apoptosis. We examined the effects of TCDD on UV-C light-induced apoptosis in primary rat hepatocytes and Huh-7 human hepatoma cells. TCDD inhibits UV-C light induced apoptosis in both cell types. This effect is seen with chromatin condensation and fragmentation and appears to be mediated by the AhR in rat hepatocytes. Apoptosis induced by UV-C light in these cells is caspase-dependent and is accompanied by alterations in apoptosis-related gene expression such as up-regulation of pro-apoptotic bcl-2 family genes like bak and bax, and a marked down-regulation of the expression of the anti-apoptotic bcl-2. TCDD treatment of irradiated hepatocytes induces the expression of some apoptosis-related genes (birc3, dad1, pycard, tnf). Up-stream apoptotic events, namely caspase-activation and caspase-substrate cleavage are not inhibited by TCDD treatment. We hypothesize that TCDD inhibits late-stage apoptotic events that lead to internucleosomal DNA fragmentation, maintaining chromosomal integrity probably in order to sustain metabolic capacity and hepatic elimination of substrates despite of an initiation of apoptosis.

Vulnerability to (+)-Methamphetamine Effects and the Relationship to Drug Disposition in Pregnant Rats during Chronic Infusion

2009-06-10

Chronic (+)-methamphetamine (METH) use during pregnancy increases the health risk for both mother and fetus. To provide insights into these risks, the relationship between changes in METH disposition and METH-induced pharmacological effects were studied in Sprague-Dawley rat dams and litters. Timed-pregnant rats (n = 5-6) were given saline or METH (5.6-17.8 mg/kg/day) by continuous sc infusion from GD7 (before organogenesis) until GD21 (0-2 days before delivery). By GD11, all rats in the 17.8 mg/kg/day group died or were sacrificed for humane reasons. There were significant (p < 0.05) dose- and gestational time-dependent decreases in maternal body weight in the 10-13.2 mg/kg/day groups, which slowly recovered to near normal by GD21. Continued METH dosing in the surviving groups did not affect the mean pups/litter weight at the end of the experiment on GD21. While maternal and fetal METH and (+)-amphetamine serum concentrations were similar on GD21, brain concentrations were significantly greater in the dams (p < 0.05). Importantly, brain-to-serum ratios in the dams were 9:1 and 3:1 in the pups. METH systemic clearance in dams significantly (p < 0.05) decreased from 52±14 ml/min/kg on GD10 to 28±6 ml/min/kg on GD21 in all dose groups, indicating late-gestational stage reductions in METH systemic clearance. Overall, these findings suggest there were two periods of increased susceptibility for dams and fetuses during chronic METH treatment. First was the period after the start of METH dosing in which neuroadaptation and tolerance to METH occurs in the adult. The second was at the end of pregnancy when METH clearance was significantly reduced.

Effects of Chronic Manganese Exposure on Glutamatergic and GABAergic Neurotransmitter Markers in the Non-Human Primate Brain

Burton, N. C., Schneider, J. S., Syversen, T., Guilarte, T. R. — 2009-06-10

The neurological sequelae of chronic Mn exposure include psychiatric, cognitive and motor deficits, suggesting the potential involvement of multiple neurotransmitter systems and brain regions. Available evidence in rodents suggests that Mn causes dysregulation of glutamatergic and GABAergic neurotransmitter systems. However, this has never been studied comprehensively in the non-human primate brain. Cynomolgus macaques were given weekly i.v. injections of 3.3-5.0mg Mn/kg, 5.0-6.7mg Mn/kg, or 8.3-10.0mg Mn/kg for 7-59 weeks. Total glutamate, glycine, and GABA concentrations were measured by HPLC with fluorescence detection in 13 brain areas in Mn-treated and control monkeys. Neurotransmitter concentrations did not change with chronic Mn exposure. Quantitative autoradiography of the N-methyl-D-aspartate receptor, the GABAa receptor, and glutamate transporters was used to assess their regional distribution. Each of these neurotransmitter receptors remained almost universally unchanged with Mn treatment. Immunohistochemical analysis of glutamine synthetase (GS) demonstrated a selective Mn-induced decrease in the globus pallidus, which could potentially alter synaptic and/or astrocytic levels of glutamate. This study shows that in non-human primates with previous documentation of Mn-induced brain pathology, the glutamatergic and GABAergic systems appear to be mostly unaffected by chronic Mn exposure with the exception of reduced GS expression in the globus pallidus.

Diabetes impairs hippocampal function via advanced-glycation-end product-mediated new neuron generation in animals with diabetes-related depression

Wang, S.-h., Sun, Z.-l., Guo, Y.-j., Yuan, Y., Yang, B.-q. — 2009-06-05

The diabetes-induced reduction of neurogenesis in hippocampal dentate and its reversal with antidepressant medications implies a potential mechanism for diabetes-related depression and cognitive decline. In the following article, the role of advanced glycation end products (AGEs) in hippocampal neurogenesis deficits in diabetic animals with depression has been further explained in the light of an in vitro study. Diabetes was induced in animals with the use of streptozotocin (55 mg/kg, i.p.), and the animals then divided into those with and those without depression-like behaviors as analyzed by behavioral tests. The AGE formation inhibitor aminoguanidine (10 mg/kg) was administrated for an additional 4 weeks. Proliferating cells, their survival, and their phenotype fate were monitored with bromodeoxyuridine labeling and confocal laser microscopy. The presence of AGE peptides was determined with the use of a flow injection assay. Animals with diabetes and depressive symptoms displayed a reduction in hippocampal neurogenesis and an elevated serum level of AGE peptides, both of which were reversed by a 4-week regimen of aminoguanidine (10 mg/kg, i.p.), which inhibits AGE formation; in addition, the depressive behaviors were improved. These findings provided in vivo evidence that diabetes impairs hippocampal function via the AGE-mediated generation of new neurons. This likely represents a putative mechanism that is responsible for diabetes-related depression and cognitive decline, and it suggests a potential approach for future research.

Activation of the aryl hydrocarbon receptor (AhR) during different critical windows in pregnancy alters mammary epithelial cell proliferation and differentiation

Lew, B. J., Collins, L. L., O'Reilly, M. A., Lawrence, B. P. — 2009-06-05

Exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes severe defects in mammary gland development and function; however, the underlying mechanism remains unclear. Alterations in epithelial cell proliferation, differentiation, and apoptosis during pregnancy-related mammary development can lead to failed lactogenesis. To determine which of these processes are affected and at what time periods, we examined proliferation, differentiation and apoptosis in mammary glands following exposure to TCDD during early, mid or throughout pregnancy. While AhR activation throughout pregnancy did not cause early involution, there was a 50% decrease in cell proliferation, which was observed as early as the 6^th day of pregnancy (DP). TCDD treatment on the day of impregnation only reduced development and proliferation in early and mid-pregnancy, followed by partial recovery by DP17. However, when AhR activation was delayed to DP7, developmental impairment was not observed in mid-pregnancy, but became evident by DP17, whereas proliferation was reduced at all times. Thus, early exposure to TCDD was neither necessary nor sufficient to cause persistent defects in lactogenesis. These varying outcomes in mammary development due to exposure at different times in pregnancy suggest there are critical windows during which AhR activation impairs mammary epithelial cell proliferation and differentiation.

Identification of Chemical Compounds that Induce HIF-1{alpha} Activity

2009-06-05

Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1 and constitutively-expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia response elements (HRE) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, while 2 compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1 inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1 activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

Distinct roles of two zebrafish AHR repressors (AHRRa and AHRRb) in embryonic development and regulating the response to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

2009-06-03

The AHR repressor (AHRR), an AHR-related bHLH-PAS protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa, AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MO) in zebrafish embryos and a zebrafish cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and TCDD toxicity. Zebrafish embryos exposed to TCDD (2 nM and 8 nM) during early development showed strong induction of CYP1A, AHRRa and AHRRb at 48 and 72 hpf. A MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to DMSO displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

The Werner Syndrome Protein Functions in Repair of Cr (VI)-induced Replication Associated DNA Damage

Liu, F.-J., Barchowsky, A., Opresko, P. L — 2009-06-01

Werner syndrome is a premature aging disorder characterized by cancer predisposition that is caused by loss of the WRN helicase/exonuclease DNA repair protein. Hexavalent chromium is an environmental carcinogen and genotoxicant that is associated with respiratory cancers and induces several forms of DNA damage, including lesions that interfere with DNA replication. Based on the evidence that WRN protein facilitates repair of stalled and collapsed replication forks, we hypothesized that WRN functions in the cellular response to and recovery from Cr(VI) induced genotoxicity and genomic instability. Here we report that human cells deficient in WRN protein are hypersensitive to Cr(VI) toxicity, and exhibit a delayed reduction in DNA breaks and stalled replication forks, indicated by H2AX foci, during recovery from Cr(VI) exposure. Cr(VI) induced WRN protein translocation from the nucleoli into nucleoplasmic foci in S-phase cells, and these foci colocalized with H2AX foci indicating WRN responds to replication associated DNA damage. As further evidence that Cr(VI) triggers stalled DNA replication, we observed Cr(VI) treatment induced an accumulation of cells in S-phase that exhibited high levels of H2AX foci. Therefore, these data demonstrate a novel role for WRN protein in cellular protection against the environmental genotoxicant Cr(VI) and further provide evidence that Cr(VI) induces DNA replicative stress which has implications for aging and cancer.

TRANSGENERATIONAL EFFECTS OF DI (2-ETHYLHEXYL) PHTHALATE IN THE MALE CRL: CD(SD) RAT: ADDED VALUE OF ASSESSING MULTIPLE OFFSPRING PER LITTER

2009-05-29

In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis and other androgen-dependent tissues. In order to define the dose-response relationship between DEHP and the Phthalate Syndrome of reproductive alterations in F1 male rats, SD rat dams were dosed by gavage from gestational day 8 to day 17 of lactation with 0, 11, 33, 100 or 300 mg/kg/d DEHP (71-93 males/dose from 12-14 litters/dose). Some of the male offspring continued to be exposed to DEHP via gavage from 18 days of age to necropsy at 63-65 days of age (PUB cohort; 16-20/dose). Remaining males were not exposed after PND17 (IUL cohort) and were necropsied after reaching full maturity. Anogenital distance, sperm counts and reproductive organ weights were reduced in F1 males in the 300 mg/kg/d group and they displayed retained nipples. In the IUL cohort, seminal vesicle weight also was reduced at 100 mg/kg/d. In contrast, serum testosterone and estradiol levels were unaffected in either the PUB or IUL cohorts at necropsy. A significant percentage of F1 males displayed one or more Phthalate Syndrome lesions at 11 mg/kg/d DEHP and above. We were able to detect effects in the lower dose groups only because we examined all the males in each litter rather than only 1 male/litter. Power calculations demonstrate how using multiple males versus one male/litter enhances the detection of the effects of DEHP. The results at 11 mg/kg/d confirm those reported from a National Toxicology Program multigenerational study (Foster et al. 2006) which reported NOAEL-LOAELs of 5 and 10 mg/kg/d DEHP respectively via the diet.

The Constitutive Active/Androstane Receptor (CAR) Facilitates Unique Phenobarbital (PB)-Induced Expression Changes of Genes Involved in Key Pathways in Precancerous Liver and Liver Tumors

Phillips, J. M., Burgoon, L. D., Goodman, J. I. — 2009-05-29

Our overall goal is to elucidate progressive changes, in expression and methylation status, of genes which play key roles in phenobarbital (PB)-induced liver tumorigenesis, with an emphasis on their potential to affect signaling through critical pathways involved in the regulation of cell growth and differentiation. PB-elicited unique expression changes of genes, including some of those identified previously as exhibiting regions of altered DNA methylation (Phillips and Goodman, 2009), were discerned in precancerous liver tissue and/or individual liver tumors from susceptible constitutive active/androstane receptor (CAR) wildtype (WT) compared to resistant CAR knockout (KO) mice. Many of these function in crucial cancer-related processes, e.g., angiogenesis, apoptosis, cell cycle, DNA methylation, Hedgehog signaling, invasion/metastasis, Notch signaling, and Wnt signaling. Furthermore, a subset of the uniquely altered genes contained CAR response elements (CAREs). This included Gadd45b, a coactivator of CAR and inhibitor of apoptosis, and 2 DNA methyltransferases (Dnmt1, Dnmt3a). The presence of CAREs in Dnmts suggests a potential direct link between PB and altered DNA methylation. The current data are juxtaposed with the effects of PB on DNA methylation and gene expression which occurred uniquely in liver tumor-prone B6C3F1 mice, as compared to the resistant C57BL/6, following 2- or 4-wk of treatment (Phillips and Goodman, 2008 and Phillips et al., 2009). Collectively, these data reveal a comprehensive view of PB-elicited molecular alterations (i.e. changes in gene expression and DNA methylation) that can facilitate hepatocarcinogenesis. Notably, candidate genes for initial "fingerprints" of early and late stages of PB-induced tumorigenesis are proposed.

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes. * II. Enzymatic Formation of Arsenylated Products Susceptible for Reduction to Arsenite by Thiols

Gregus, Z., Roos, G., Geerlings, P., Nemeti, B. — 2009-05-28

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiothreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e. ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT ~ DMPS > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.

Benzene-induced hematopoietic neoplasms including myeloid leukemia in Trp53-deficient C57BL/6 and C3H/He mice

2009-05-28

This research focused on three major questions regarding benzene-induced hematopoietic neoplasms (HPNs). First, why are HPNs induced equivocally and at only threshold level with low-dose benzene exposure despite the significant genotoxicity of benzene even at low-doses both in experiments and epidemiology? Second, why is there no linear increase in incidence at high-dose exposure despite a lower acute toxicity (LD₅₀ > 1000 mg/kg body weight; WHO, 2003) ? Third, why are particular myeloid leukemias (AMLs) not commonly observed in mice even though AMLs are frequently observed in human cases of occupational exposure to benzene? In this study, we hypothesized that the threshold-like equivocal induction of HPNs at low-dose benzene exposure is based on DNA-repair potential in wild-type mice, and that the limited increase in HPNs at a high-dose exposure is due to excessive apoptosis in wild-type mice. To determine whether Trp53-deficiency satisfies the above hypotheses by eliminating or reducing DNA repair and by allowing cells to escape apoptosis, we evaluated the incidence of benzene-induced HPNs in Trp53-deficient C57BL/6 mice with specific regard to AMLs. We also used C3H/He mice, AML-prone, with Trp53-deficiency, to explore whether a higher incidence of AMLs on benzene exposure might explain the above human-murine differences. As a result, heterozygous Trp53-deficient mice of both strains showed a non-threshold response of the incidence of HPNs at the lower dose, whereas both strains showed an increasing HPN incidence up to 100% with increasing in benzene exposure dose; including AMLs, that developed 38% of heterozygous Trp53-deficient C3H/He mice compared to only 9% of wild-type mice exposed to the high dose. The detection of AMLs in heterozygous Trp53-deficient mice, even in the C57BL/6 strain, implies that benzene may be a potent inducer of AMLs also in mice with some strain differences.

Introducing the 'TCDD Inducible AhR-Nrf2 Gene Battery'

Yeager, R. L., Reisman, S. A., Aleksunes, L. M., Klaassen, C. D. — 2009-05-27

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H: quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the ‘AhR gene battery’. However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 µg/kg, i.p.) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid synthesizing gene Ugdh, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 (MGst1) in WT mice but not Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes. I. The Role of Arsenolysis

Nemeti, B., Gregus, Z. — 2009-05-27

Several mammalian enzymes catalyzing the phosphorolytic-arsenolytic cleavage of their substrates (thus yielding arsenylated metabolites) have been shown to facilitate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in presence of their substrate and a thiol. These include purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycogen phosphorylase-a (GPa). In this work, we tested further enzymes, the bacterial phosphotransacetylases (PTA) and PNP, for AsV reduction. The PTAs, which arsenolytically cleave acetyl-CoA producing acetyl-arsenate, were compared to GAPDH, which can also form acetyl-arsenate by arsenolysis of its non-physiological substrate, acetyl-phosphate. As these enzymes also mediated AsV reduction, we can assert that facilitation of thiol-dependent AsV reduction may be a general property of enzymes that catalyze phosphorolytic-arsenolytic reactions. Because with all such enzymes arsenolysis is obligatory for AsV reduction, we analyzed the relationship between these two processes in presence of various thiol compounds, using PNP. While no thiol influenced the rate of PNP-catalyzed arsenolysis, all enhanced the PNP-mediated AsV reduction, albeit differentially. Furthermore, the relative capacity of thiols to support AsV reduction mediated by PNP, GPa, PTA, and GAPDH apparently depended on the type of arsenylated metabolites (i.e., arsenate ester or anhydride) produced by these enzymes. Importantly, AsV reduction by both acetyl-arsenate-producing enzymes (i.e., PTA and GAPDH) exhibited striking similarities in responsiveness to various thiols, thus highlighting the role of arsenylated metabolite formation. This observation, together with the finding that PNP-mediated AsV reduction lags behind the PNP-catalyzed arsenolysis lead to the hypothesis that arsenolytic enzymes promote reduction of AsV by forming arsenylated metabolites which are more reducible to AsIII by thiols than inorganic AsV. This hypothesis is evaluated in the adjoining paper.

Protein Profiles in Zebrafish (Danio rerio) Embryos Exposed to PFOS

Shi, X., Yeung, L. W. Y., Lam, P. K. S., Wu, R. S. S., Zhou, B. — 2009-05-27

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and in wildlife, and it has the potential for developmental toxicity. However, the molecular mechanisms that lead to these toxic effects are not well-known. In the present study, proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebrafish embryos exposed to 0.5 mg/L PFOS until 192 hours post-fertilization. Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that 69 proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels (more than 2-fold difference). Of the 69 spots corresponding to the proteins with altered expression, 38 were selected and subjected to matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 18 proteins were identified in this analysis. These proteins can be categorized into diverse functional classes such as detoxification, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. Overall, proteomic analysis using zebrafish embryos serves as an in vivo model in environmental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity.

"A 21st century paradigm for evaluating the health hazards of nanoscale materials?"

Walker, N. J., Bucher, J. R. — 2009-05-25

Over the past 5 years we have seen an increase in the attention focused on the assessment of the potential health risk posed by nanoscale materials. The diversity of these materials with respect to size, composition and surface properties, and the rapid pace of their development and commercialization, poses significant challenges to traditional toxicity testing paradigms. At the same time the potential use of new high throughput "predictive "toxicity" strategies, such as that envisioned in the recent NRC report "Toxicity Testing in the 21st Century", have emerged as possible solutions to deal with the issue of how to assess the safety of the thousands of chemicals to which humans are potentially exposed. In this forum article we discuss how in some respects, the emergence of diverse engineered nanomaterials offers a tailor-made test case for the application of a new paradigm for assessing human heath risks. However while this approach may have merit in the study of some specific nanomaterials, this approach does not consider the complexity involved in utilizing in vitro cell culture toxicology methods to evaluate the potential hazard of the wide array of current and future engineered nanomaterials.

Mode of Action for Reproductive and Hepatic Toxicity Inferred from a Genomic Study of Triazole Antifungals

Goetz, A. K., Dix, D. J. — 2009-05-25

The mode of action for the reproductive toxicity of some triazole antifungals has been characterized as an increase in serum testosterone and hepatic response, and reduced insemination and fertility indices. In order to refine our mechanistic understanding of these potential modes of action, gene expression profiling was conducted on liver and testis from male Wistar Han IGS rats exposed to myclobutanil (500, 2000 ppm), propiconazole (500, 2500 ppm), or triadimefon (500, 1800 ppm) from gestation day six (GD6) to postnatal day 92 (PND92). Gene expression profiles indicated that all three triazoles significantly perturbed the fatty acid, steroid, and xenobiotic metabolism pathways in the male rat liver. In addition, triadimefon modulated expression of genes in the liver from the sterol biosynthesis pathway. While expression of individual genes were affected, there were no common pathways modulated by all three triazoles in the testis. The pathways identified in the liver included numerous genes involved in phase I-III metabolism (Aldh1a1, Cyp1a1, Cyp2b2, Cyp3a1, Cyp3a2, Slco1a4, Udpgtr2), fatty acid metabolism (Cyp4a10, Pcx, Ppap2b), and steroid metabolism (Ugt1a1, Ugt2a1) for which expression was altered by the triazoles. These differentially expressed genes form part of a network involving lipid, sterol and steroid homeostatic pathways regulated by the constitutive androstane (CAR), pregnane X (PXR), peroxisome proliferator-activated alpha (PPAR), and other nuclear receptors in liver. These relatively high dose and long term exposures to triazole antifungals appeared to perturb fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways. These toxicogenomic effects describe a plausible series of key events contributing to the disruption in steroid homeostasis and reproductive toxicity of select triazole antifungals.

Methylmercury speciation influences brain gene expression and behaviour in gestationally-exposed mice pups

2009-05-22

The greatest source of human exposure to methylmercury (MeHg) is the diet, in particular the consumption of seafood. To investigate the importance of dietary MeHg speciation on neurotoxicity, balb/c mice dams were exposed to MeHgCys (the naturally-occurring salt) and MeHgCl (the laboratory salt), at concentrations up to 4.5 mg kg^–1, for eleven weeks (inclusive of three weeks gestational and two weeks post-partum exposure). Impacts of developmental exposure were assessed in their offspring by monitoring transcriptomic (brain gene expression via microarray and quantitative PCR), tissue mercury (Hg) accumulation, and neurobehavioural endpoints. There were no differences in tissue Hg accumulation between the two forms of MeHg presented, but differences in pup behaviour and gene expression endpoints were noted. For example, MeHgCl, but not MeHgCys, impaired pup activity in an open field assessment. Similar impacts of MeHgCl were noted in adults. A total of 131 genes were differentially-regulated in pup brains following maternal exposure to MeHg, 50 of which were specific to MeHgCys and 35 specific to MeHgCl. Regulated genes were significantly enriched for several annotation categories including metal/zinc-binding and transcription regulation. In contrast few antioxidant genes were differentially-regulated. This analysis provided insight into mechanisms by which MeHg may impair cellular processes in addition to behavioural impairments such as those associated with learning and memory. The results show differences between the toxic impacts of MeHg species, and also highlight the potential utility of an integrated approach incorporating gene expression with behavioural endpoints.

Characterization and interlaboratory comparison of a gene expression signature for differentiating genotoxic mechanisms

2009-05-22

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, since chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multi-laboratory project coordinated by the HESI Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene-set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

Oxidative stress induces parallel autophagy and mitochondria dysfunction in human glioma U251 cells

Zhang, H., Kong, X., Kang, J., Su, J., Li, Y., Zhong, J., Sun, L. — 2009-05-18

Accumulation of reactive oxygen species (ROS) such as Hydrogen peroxide (H₂O₂), is an oxidative stress response induced various defense mechanisms or program cell death (PCD). As one of the major types of program cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. To date, however, the exact mechanism by which reactive oxygen species regulate autophagy is still poorly understood. Thus the purpose of this study was to elucidate how H₂O₂ exerts its cytotoxic effects on malignant glioma U251 cells, and to uncover the molecular mechanism that might be involved. Here we show that H₂O₂ induced autophagy and apoptosis in U251 cells, is mediated though the Beclin 1 and Akt/mTOR pathways. Accumulation of ROS leads to changes in mitochondrial permeability with loss of mitochondrial membrane potential, and disruption of mitochondrial dynamics at a transcriptional level of fission and fusion. Overexpression of cellular Bcl-2 partially inhibited autophagy though both the Beclin 1 and Akt/mTOR pathways, and led to recovery of mitochondrial dynamics. When autophagy was prevented at an early stage by 3-methyladenine, apoptosis significantly increased. Our data provide the first evidence that H₂O₂ induces autophagy through interference with the Beclin 1 and Akt/mTOR signaling pathways, and is regulated by the anti-apoptotic gene Bcl-2 in glioma U251 cells.

Use of physiologically based biokinetic (PBBK) modeling to study estragole bioactivation and detoxification in humans as compared to male rats.

2009-05-15

The extent of bioactivation of the herbal constituent estragole to its ultimate carcinogenic metabolite, 1'-sulfooxyestragole depends on the relative levels of both bioactivation and detoxification pathways. The present study investigated the kinetics of the metabolic reactions of both estragole and its proximate carcinogenic metabolite 1'-hydroxyestragole in humans in incubations with relevant tissue fractions. Based on the kinetic data obtained a PBBK model for estragole in human was defined to predict the relative extent of bioactivation and detoxification at different dose levels of estragole. The outcomes of the model were subsequently compared to those previously predicted by a PBBK model for estragole in male rat to evaluate the occurrence of species differences in metabolic activation. The results obtained reveal that formation of 1'-oxoestragole, which represents a minor metabolic route for 1'-hydroxyestragole in rat, is the main detoxification pathway of 1'-hydroxyestragole in humans. Due to a high level of this 1'-hydroxyestragole oxidation pathway in human liver, the predicted species differences in formation of 1'-sulfooxyestragole remain relatively low, with the predicted formation of 1'-sulfooxyestragole being 2-fold higher in human compared to male rat, even though the formation of its precursor 1'-hydroxyestragole was predicted to be 4–fold higher in human. Overall, it is concluded that in spite of significant differences in the relative extent of different metabolic pathways between human and male rat there is a minor influence of species differences on the ultimate overall bioactivation of estragole to 1'-sulfooxyestragole.

Absence of carcinogenic response to multi-wall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat

Muller, J., Delos, M., Panin, N., Rabolli, V., Huaux, F., Lison, D. — 2009-05-08

Toxicological investigations of carbon nanotubes have shown that they can induce pulmonary toxicity, and similarities with asbestos fibers have been suggested. We previously reported that multi-wall carbon nanotubes (MWCNT) induced lung inflammation, granulomas and fibrotic reactions. The same MWCNT also caused mutations in epithelial cells in vitro and in vivo. These inflammatory and genotoxic activities were related to the presence of defects in the structure of the nanotubes. In view of the strong links between inflammation, mutations and cancer, these observations prompted us to explore the carcinogenic potential of these MWCNT in the peritoneal cavity of rats.

The incidence of mesothelioma and other tumors was recorded in 3 groups of 50 male Wistar rats injected intraperitoneally with a single dose of MWCNT with defects (2 or 20 mg/animal) and MWCNT without defects (20 mg/animal). Two additional groups of 26 rats were used as positive (2 mg UICC crocidolite /animal) and vehicle controls.

After 24 months, while crocidolite induced a clear carcinogenic response (34.6% animals with mesothelioma vs 3.8% in vehicle controls), MWCNT with or without structural defects did not induce mesothelioma in this bioassay (4, 0 or 6%, respectively). The incidence of tumors other than mesothelioma was not significantly increased across the groups.

The initial hypothesis of a contrasting carcinogenic activity between MWCNT with and without defects could not be verified in this bioassay. We discuss the possible reasons for this absence of carcinogenic response, including the length of the MWCNT tested (<1 µm on average), the absence of a sustained inflammatory reaction to MWCNT, and the capacity of these MWCNT to quench free radicals.

Dominant Role of Orai1 with STIM1 on the Cytosolic Entry and Cytotoxicity of Lead Ions

Chiu, T.-Y., Teng, H.-C., Huang, P.-C., Kao, F.-J., Yang, D.-M. — 2009-05-08

Pb²⁺ ions cause severe damages to living cells. In particular, our previous study showed that the Orai-STIM1-formed store-operated Ca²⁺ channels (SOCs) allow Pb²⁺ entry. In relation to this, the present study investigates the molecular gating mechanism of Pb²⁺ entry by Orai1 with STIM1, as well as the resulting cytotoxicity on human embryonic kidney HEK293 cells. The store-operated Ca²⁺ entry (SOCE, activity of SOCs) and Pb²⁺ entry were measured using the fura-2 imaging method and indo-1 quenching strategy, as well as through an atomic absorption spectrophotometer. The results of RT-PCR, Western blot, fast confocal, and fluorescent lifetime imaging microscopy indicated the endogenous expression of Orai1 and STIM1 in HEK cells and the functional interaction between these two proteins during SOCE. Both SOCE and Pb²⁺ entry largely increased when Orai1 and STIM1 were overexpressed (3- and 1.64-folds, respectively) compared with non-fluorescent cells, and they were significantly attenuated when the E106Q mutation of Orail with STIM1 was co-transfected (6- and 2.25-folds decrease, respectively) compared with Orai1-STIM1 co-expressed cells. The ion gating for Pb²⁺ could be governed by the E106 region of Orai1. After sorting and subsequent cultures, the Orai1-STIM1 positive expressed cells behaved more sensitively to Pb²⁺ than the Orai1-STIM1 negative cells. In summary, the data suggest that Orai1, together with STIM1, plays a critical role in Pb²⁺ entry and the toxicity of Pb²⁺.

Structure-Activity Relationships and Human Relevance for Perfluoroalkyl Acid-induced Transcriptional Activation of Peroxisome Proliferation in Liver Cell Cultures

Bjork, J. A., Wallace, K. B. — 2009-04-30

Perfluoroalkyl acids (PFAAs) are widely distributed and environmentally persistent agents whose potential toxicity isn't yet fully characterized. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) elicit a number of potential toxicities in rodents, the most prevalent of which are governed by activation of the peroxisome proliferators activator receptor alpha (PPAR). The purpose of this investigation was two-fold: 1) conduct a structure-activity relationship (SAR) study of the transcriptional activation of peroxisome proliferation in primary rat liver cell cultures for PFAA-related carboxylic and sulfonic acids of varying carbon chain length and, 2) explore whether this activity can be translated to human liver cells in culture. Exposure to PFOA caused a dose-dependent stimulation of the expression of Acox, Cte/Acot1 and Cyp4a1/11, transcripts that are indicative of peroxisome proliferation, in primary rat hepatocytes. PFOA concentrations of 30µM and above caused cell injury characterized by the expression of Ddit3. PFBA, on the other hand, stimulated Acox, Cte/Acot1 and Cyp4a1/11 gene expression in primary rat hepatocytes only at concentrations of 100µM and above. Neither PFOA nor PFBA at concentrations up to 200µM stimulated PPAR-related gene expression in either primary or HepG2 human liver cells. These data demonstrate that 1) PFFAs cause a concentration and chain-length dependent increase in expression of gene targets related to cell injury and PPAR activation in primary rat hepatocytes, 2) the sulfonates are less potent than the corresponding carboxylates in stimulating PPAR-related gene expression in rat hepatocytes, and 3) stimulation of PPAR-mediated gene transcription is a mechanism that is not shared by human liver cells, adding further substantiation that PPAR-dependent liver toxicity in rodents does not extrapolate to assessing human health concerns.

Ambient Particulates Alter Vascular Function through Induction of Reactive Oxygen and Nitrogen Species

2009-04-15

Background.

Previous studies have shown a link between inhaled particulate matter (PM) exposure in urban areas and susceptibility to cardiovascular diseases. While an oxidative stress pathway is strongly implicated, the locus of generation of reactive oxygen species (ROS) and the mechanisms by which these radicals exert their effects remain to be characterized.

Objective.

To test the hypothesis that exposure to environmentally relevant inhaled concentrated ambient particulate matter (CAPs) enhances atherosclerosis through induction of vascular ROS and reactive nitrogen species.

Method.

High-fat chow fed apolipoprotein E^–/– mice were exposed to CAPs of less than 2.5 µm (PM_2.5) or filtered air (FA), for 6h/d, 5d/wk, for 4 months in Manhattan, NY. Atherosclerotic lesions were analyzed histomorphometricly. Vascular reactivity, superoxide generation, mRNA expression of NADPH oxidase subunits, iNOS, eNOS, and GTP cyclohydrolase I were also assessed.

Results.

Manhattan PM_2.5 CAPs were characterized by high concentrations of organic and elemental carbon (OC/EC). Analysis of vascular responses revealed significantly decreased phenylephrine constriction in CAPs-exposed mice, which was restored by a soluble guanine cyclase inhibitor ODQ. Vascular relaxation to A23187, but not to acetylcholine, was attenuated in CAPs mice. Aortic expression of NADPH oxidase sub-units (p47^phox and rac1) and iNOS were markedly increased, paralleled by increases in superoxide generation and extensive protein nitration in the aorta. The composite plaque area of thoracic aorta was significantly increased with pronounced macrophage infiltration and lipid deposition in the CAPs mice.

Conclusions.

CAPs exposure in Manhattan alters vasomotor tone and enhances atherosclerosis through NADPH oxidase dependent pathways.

OGG1 and MYH are involved in the incision of trivalent arsenical-induced DNA adducts

Pu, Y.-S., Jan, K.-Y., Wang, T.-C., Wang, A. S. S., Gurr, J.-R. — 2006-11-29

Since trivalent arsenicals are known to induce oxidative DNA damage in human cells, we asked if they induce other types of DNA damage and how these DNA damages are repaired. Treatment of human promyelocytic leukemia NB4 cells with 0.5 µM As₂O₃ for 30 min induced no DNA breaks, as analyzed by a standard comet assay. However, breaks were detected if these cells were then digested with endonuclease III (EnIII), formamidopyrimidine-DNA glycosylase (Fpg), or a nuclear extract (NE) of NB4 cells. Using either H₂O₂-Fe treated nuclei or As₂O₃-treated cells, digestion with either NE or EnIII+Fpg generated the same amount of breaks, and subsequent treatment with EnIII+Fpg resulted in no increase in breaks in NE-digested cells and vice versa. The human cell lines, defective in nucleotide excision protein, such as XPA, XPD, and XPG, excised UVC-induced adducts less rapidly than normal fibroblasts, but excised As₂O₃-adducts at the same rate as the normal cells. Immunodepletion of the NE with antibody against OGG1 or MYH decreased the incision of DNA adducts induced by As₂O₃, NaAsO₂, monomethylarsonic acid, and dimethylarsinic acid, while antibodies against XPA, XPB, XPD, XPF, or XPG, did not. These results suggest that these trivalent arsenicals induce the formation of only oxidative DNA adducts and that OGG1 and MYH are involved in these incision processes.