Alcohol and MDMA (ecstasy) are frequently co-abused, but recent findings indicate a harmful drug interaction between these two agents. In our previous study, we showed that MDMA exposure inhibits the activity of the acetaldehyde (ACH) metabolizing enzyme, aldehyde dehydrogenase2 (ALDH2). Based on this finding, we hypothesized that the co-administration of MDMA and ethanol would reduce the metabolism of ACH and result in increased accumulation of ACH. Rats were treated with MDMA or vehicle and then administered a single dose of ethanol. Liver ALDH2 activity decreased by 35% in the MDMA-treated rats compared to control rats. The peak concentration and the area under the concentration versus time curve of plasma ACH was 31% and 59% higher, respectively, in the MDMA-ethanol group compared to the ethanol-only group. In addition, the MDMA-ethanol group had 80% higher plasma transaminase levels than the ethanol-only group, indicating greater hepatocellular damage. Our results not only support a drug interaction between MDMA and ethanol but a novel underlying mechanism for the interaction.

Inorganic arsenic, an early life carcinogen in humans and mice, can initiate lesions promotable by other agents in later life. The biomethylation product of arsenic, dimethylarsinic acid (DMA), is a multi-site tumor promoter. Thus, pregnant CD1 mice were given drinking water (0 or 85 ppm arsenic) from gestation day 8 to 18 and after weaning male offspring received DMA (0 or 200 ppm; drinking water) for up to 2 years. No renal tumors occurred in controls or DMA alone treated mice while gestational arsenic exposure plus later DMA induced a significant renal tumor incidence of 17% (primarily renal cell carcinoma). Arsenic plus DMA or arsenic alone also increased renal hyperplasia over control but DMA alone did not. Arsenic alone, DMA alone and arsenic plus DMA all induced urinary bladder hyperplasia (33–35%) versus control (2%). Compared to control (6%), arsenic alone tripled hepatocellular carcinoma (20%), and arsenic plus DMA doubled this rate again (43%), but DMA alone had no effect. DMA alone, arsenic alone, and arsenic plus DMA increased lung adenocarcinomas and adrenal adenomas versus control. Overall, DMA in adulthood promoted tumors/lesions initiated by prenatal arsenic in the kidney and liver, but acted independently in the urinary bladder, lung and adrenal.

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.

Human exposure to particulate matter (PM) elicits a variety of responses on the cardiovascular system through both direct and indirect pathways. Indirect effects of PM on the cardiovascular system are mediated through the autonomic nervous system, which controls heart rate variability, and inflammatory responses, which augment acute cardiovascular events and atherosclerosis. Recent research demonstrates that PM also affects the cardiovascular system directly by entry into the systemic circulation. This process causes myocardial dysfunction through mechanisms of reactive oxygen species production, calcium ion interference, and vascular dysfunction. In this review, we will present key evidence in both the direct and indirect pathways, suggest clinical applications of the current literature, and recommend directions for future research.

Despite data linking amphibole asbestos exposure with production of autoantibodies, the role of autoantibodies in subsequent disease is unknown. Residents of Libby, Montana have experienced significant exposure to amphibole asbestos due to the mining of asbestos-contaminated vermiculite near the community over several decades. This population predominantly exhibits pleural disease, and an autoimmune-like disorder that has yet to be well defined. This study sought to determine whether autoantibodies from asbestos-exposed subjects were associated with pleural lesions. Serum samples of subjects from Libby were evaluated for anti-nuclear antibodies (ANA) and mesothelial cell autoantibodies (MCAA) using cell based ELISA. The presence of radiographic abnormalities detected during the time frame of serum collection was determined from screening records. In accord with previous studies, 61.3% (76/124) of the Libby samples were ANA positive, a frequency much higher than expected for a healthy population. The odds of having pleural or interstitial abnormalities in Libby was nearly 3.55 times greater for individuals that tested positive for ANA compared with individuals negative for ANA (p =0.004). MCAA were also detected at a strikingly high frequency (18.5%; 23/124) in samples from Libby. Individuals with MCAA had 4.9 times the risk of having pleural abnormalities compared to MCAA-negative subjects (p=0.044). In conclusion, ANA and MCAA were elevated in a study population that was known to have chronic exposure to asbestos, and these autoantibodies were associated with pleural abnormalities, the predominant finding in the asbestos-exposed population of Libby. Additional research is needed to determine the role these autoantibodies may play in pulmonary disease.

Dioxins are widespread environmental contaminants that induce the carcinogen-activating enzyme, cytochrome P450 1A1 (CYP1A1) through an aryl hydrocarbon receptor (AhR)-dependent mechanism. We previously demonstrated that harmine inhibits the dioxin-mediated induction of Cyp1a1 activity in murine hepatoma cells. Therefore, the aim of this study is to determine the effect of harmine and its main metabolite, harmol, on the dioxin-mediated induction of CYP1A1 in human HepG2 and murine Hepa 1c1c7 hepatoma cells. Our results showed that harmine and harmol significantly inhibited the dioxin-mediated induction of CYP1A1 at mRNA, protein, and activity levels in a concentration-dependent manner in human and murine hepatoma cells. Moreover, harmine and harmol inhibited the AhR-dependent luciferase activity and the activation and transformation of AhR using the electrophoretic mobility shift assay. In addition, harmine and harmol displaced [3H]TCDD in the competitive ligand binding assay. At posttranslational level, both harmine and harmol decreased the protein stability of CYP1A1, suggesting that posttranslational mechanism is involved. Furthermore, we demonstrated that the underlying mechanisms of the posttranslational modifications of both compounds involve ubiquitin-proteasomal pathway and direct inhibitory effects of CYP1A1 enzyme. We concluded that harmine and its metabolite, harmol, are new inhibitors of dioxin-mediated effects.

Increased levels of inducible nitric oxide synthase (iNOS) during cardiac stress such as ischemia-reperfusion, sepsis and hypertension may display both beneficial and detrimental roles in cardiac contractile performance. However, the precise role of iNOS in the maintenance of cardiac contractile function remains elusive. This study was designed to determine the impact of chronic iNOS inhibition on cardiac contractile function and the underlying mechanism involved with a special focus on the NO downstream signaling molecule Akt. Male C57 or Akt2 knockout [Akt2(−/−)] mice were injected with the specific iNOS inhibitor 1400W (2 mg/kg/d) or saline for 7 days. Both 1400W and Akt2 knockout dampened glucose and insulin tolerance without additive effects. Treatment of 1400W decreased heart and liver weights as well as cardiomyocyte cross-sectional area in C57 but not Akt2 knockout mice. 1400W but not Akt2 knockout compromised cardiomyocyte mechanical properties including decreased peak shortening and maximal velocity of shortening/relengthening, prolonged relengthening duration, reduced intracellular Ca2+ release and decay rate, the effects of which were ablated or attenuated by Akt2 knockout. Akt2 knockout but not 1400W increased the levels of intracellular Ca2+ regulatory proteins including SERCA2a and phospholamban phosphorylation. 1400W reduced the level of anti-apoptotic protein Bcl-2, the effect of which was unaffected by Akt2 knockout. Neither 1400W nor Akt2 knockout significantly affected ER stress, autophagy, the post-insulin receptor signaling Akt, GSK3β and AMPK, as well as the stress signaling IΚB, JNK, ERK and p38 with the exception of elevated IΚB phosphorylation with jointed effect of 1400W and Akt2 knockout. Taken together, these data indicated that an essential role of iNOS in the maintenance of cardiac morphology and function possibly through an Akt2-dependent mechanism.

Esophageal cancer has been associated with tobacco and alcohol consumption, gastric reflux, exposure to nitrosamines from food or other environmental sources, and diets lacking folate. Susceptibility to esophageal cancer may be modified by functional polymorphisms in genes along the folate metabolic pathway, such as methylenetetrahydrofolate reductase (MTHFR). The C677T polymorphism is the most common functional variant, leading to a reduction in enzyme activity. We report a pooled analysis of 5 studies on the association of MTHFR C677T polymorphism and esophageal cancer, including 725 cases and 1531 controls. A significant association between the MTHFR 677 TT genotype and esophageal cancer was observed (OR = 2.63, 95% CI: 1.75–3.94), although there was significant heterogeneity between studies. A sensitivity analysis excluded one study; the association between TT genotype and esophageal cancer was still present, although of reduced magnitude (OR = 1.57, 95% CI: 0.96–2.56). A significant interaction between smoking and TT genotype on esophageal cancer risk was observed, while no interaction was observed between alcohol consumption and genotype.

Although BRCA1 is the most prevalent genetic factor in breast cancer, the pathologic mechanism of tumorigenesis caused by its deficiency has not been elucidated. We have previously demonstrated that BRCA1 can modulate responses to xenobiotic stress by regulating expression of genes involved in metabolic activation, detoxification and antioxidant reactions. In this study, we examined whether BRCA1 deficiency is more vulnerable to xenobiotic stress by employing an in vitro cell model system. Benzo[a]pyrene (B[a]P), used as a xenobiotic insult, increased intracellular reactive oxygen species (ROS) levels in breast epithelial cells. Accumulation of ROS upon B[a]P exposure was significantly augmented by abrogation of BRCA1 compared to the control. Overexpression of Nrf2 in BRCA1 deficient cells reduced elevated ROS to the control levels. Bioactive food components such as sulforaphane (SFN) and resveratrol (RSV) significantly reduced B[a]P-induced ROS accumulation regardless of BRCA1 presence. In addition, these bioactive food components increased Nrf2 levels and Nrf2 transcriptional activity, which led to attenuation of B[a]P-induced DNA damages. Likewise, incubation with bioactive food components reduced B[a]P-mediated DNA damage in BRCA1 deficient cells. In conclusion, we demonstrated that the lack of BRCA1 renders cells more susceptible to ROS-induced DNA damage, which may eventually result in tumorigenesis, and that administration of Nrf2-activating bioactive food components can reduce those risks.

Humans are chronically exposed to the plasticizer, Bisphenol A (BPA) that can adversely affect the normal hormonal regulation of cellular functions by mimicking the actions of estrogen. This biological response to BPA may vary according to an individual’s genetic characteristics (e.g., BRCA1 mutations or deletion). In this study, both cell culture and mouse models were used to elucidate whether the loss of BRCA1 function could affect BPA-mediated cell proliferation. In studies using BPA levels comparable to human exposures, we found that loss of BRCA1 enhances BPA-induced cell proliferation in both systems. In vitro, we found that loss of BRCA1 enhances BPA-induced ERα signaling. In vivo, we found that BPA administration stimulates mammary gland epithelial tissue/cell proliferation leading to hyperplasia in Brca1 mutant mice compared to wild- type control mice. These results suggest that the biological responses in BRCA1-deficient cells may depend on environmental exposures, specifically BPA.

This study was designed to analyze the effect of environmental oxidative stress on human placental monooxygenases, glutathione S-transferase (GST) activity and polycyclic aromatic hydrocarbon (PAH)–DNA adducts in human term placentas from radioactivity-contaminated and chemically-polluted areas of the Ukraine and Belarus, and to compare these biomarkers to the newborn’s general health status. Placental PAH–DNA adduct formation, GST activity, 7-ethoxycoumarin O-deethylase (ECOD) activity, and thiobarbituric reactive substances (TBARS), an index of lipid peroxidation, were measured in groups of women exposed to different levels of radioactivity and PAH pollution. The in vitro metabolism data, obtained from 143 human placental samples at term, were compared to indices of maternal and newborn health. The highest ECOD activity was recorded in placentas obtained from chemically-polluted areas and a radioactivity-contaminated area; the ECOD activity was 7-fold and 2-fold higher compared to the region considered to be “clean”. Newborns with the most compromised health status displayed the greatest down-regulation of GST activity (144–162 mU mg protein−1 vs. 258–395 mU mg protein−1), enhanced ECOD activity and the highest level of PAH–DNA adduct formation. The highest level of TBARS was observed in women exposed to the highest levels of radiation. The efficiency of placental detoxification negatively correlated with maternal age and the health status of the newborn. Environmental oxidative stress was related to an increase in anemia, threatened abortions, toxemia, fetal hypoxia, spontaneous abortions and fetal hypotrophy. Our data suggest that chemically- or radioactivity-induced oxidative stress enhance cytochrome P450-mediated enzymatic activities potentially resulting in increased formation of reactive metabolites. The activity of GSH-transferase is not enhanced. This imbalance in detoxification capacity can be measured as increased production of PAH–DNA adducts, decreased lipid peroxidation and compromised fetal health.

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation.

MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to 1) determine if ANP is released following MDMA administration, and 2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60 min following MDMA (40 mg/kg, sc) administration in 16 male Sprague-Dawley rats. A robust increase in ANP was seen within ten min of MDMA administration. ANP levels returned to baseline at 20 min and then gradually rose over the 60 min monitoring period. The administration of Anantin (40 mg, ip), 15 min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA.

A number of studies have demonstrated that co-exposure to low levels of melamine and cyanuric acid elicits renal toxicity due to the formation of melamine cyanurate crystals in the kidney nephrons. In this work, we investigated if co-exposure of rats to these compounds leads to alterations in the expression of the genes encoding kidney injury molecule 1 (KIM-1), metallopeptidase inhibitor 1 (TIMP1), clusterin, osteopontin, and neutrophil gelatinase-associated lipocalin/lipocalin 2 (NGAL), which have been proposed as urinary biomarkers for nephrotoxicity. Six-week-old male and female F344 rats were fed ad libitum a diet fortified with 0 (control), 7, 23, 69, 229, or 694 ppm melamine and cyanuric acid (co-exposure groups), 1388 ppm melamine, or 1388 ppm cyanuric acid for seven days. Histopathology and clinical chemistry examination indicated marked toxicity only in the animals exposed to the two highest combined doses of melamine and cyanuric acid. Consistent with these observations, quantitative real-time polymerase chain reaction analysis of kidney tissue indicated increased expression of all genes analyzed relative to the control in both male and female rats fed daily with 229 or 694 ppm melamine and cyanuric acid. Exposure to lower levels of both compounds or to the individual compounds did not induce gene expression changes. These data indicate that quantifying the expression levels of the selected biomarker genes constitutes a useful endpoint to assess the combined toxicity of melamine and cyanuric acid in both male and female rats.

Poisoning by nerve agents via the percutaneous (p.c.) route is an issue because the slow absorption of agent could result in poisoning which outlasts the protection provided by conventional pharmacological therapy. The bioscavenger approach is based on the concept of binding nerve agent in the bloodstream, thus preventing nerve agent from reaching the target tissues and inhibiting acetylcholinesterase activity. One bioscavenger that has been extensively studied is human butyrylcholinesterase (huBuChE). Protexia® is a pegylated form of recombinant huBuChE. We used a guinea-pig model of p.c. nerve agent poisoning, using an implanted telemetry system to collect physiological data. Guinea-pigs were poisoned with the nerve agent VX (0.74 mg/kg) (~2.5×LD50). Two hours following VX exposure, Protexia (72 mg/kg) or saline control was administered intramuscularly. All guinea-pigs treated with Protexia (n=8) survived, compared to no survivors in a saline-treated control group (n=8). Survival following VX and Protexia treatment was associated with minimal incapacitation and observable signs of poisoning, and the mitigation or prevention of the detrimental physiological changes (e.g. seizure, bradycardia and hypothermia) observed in control animals. The opportunity for post-exposure treatment may have utility in both civilian and military scenarios, and this is a promising indication for the use of a bioscavenger.

An ex-vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its kcat/Km value for GF. This protocol thus provides a rapid and reliable ex-vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.

Highlights

► The metabolism of deoxynivalenol-3-glucoside (D3G) in rats was studied. ► Urine and feces were analyzed by a validated LC–MS/MS biomarker method. ► D3G was readily hydrolyzed to deoxynivalenol (DON) during digestion. ► Most D3G was metabolized by the gut microbiota and recovered in feces. ► D3G is of considerably lower toxicological relevance than DON, at least in rats.

Deoxynivalenol-3-β-d-glucoside (D3G), a plant metabolite of the Fusarium mycotoxin deoxynivalenol (DON), might be hydrolyzed in the digestive tract of mammals, thus contributing to the total dietary DON exposure of individuals. Yet, D3G has not been considered in regulatory limits set for DON for foodstuffs due to the lack of in vivo data. The aim of our study was to evaluate whether D3G is reactivated in vivo by investigation of its metabolism in rats. Six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight (b.w.)) and the equimolar amount of D3G (3.1 mg/kg b.w.) by gavage on day 1, 8 and 15, respectively. Urine and feces were collected for 48 h and analyzed for D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) by a validated LC–tandem mass spectrometry (MS/MS) based biomarker method. After administration of D3G, only 3.7 ± 0.7% of the given dose were found in urine in the form of analyzed analytes, compared to 14.9 ± 5.0% after administration of DON, and only 0.3 ± 0.1% were detected in the form of urinary D3G. The majority of administered D3G was recovered as DON and DOM-1 in feces. These results suggest that D3G is little bioavailable, hydrolyzed to DON during digestion, and partially converted to DOM-1 and DON-GlcA prior to excretion. Our data indicate that D3G is of considerably lower toxicological relevance than DON, at least in rats.

Bifunctional alkyalating agent, Sulfur mustard (SM)-caused cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 or 4 mg CEES for 9–48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in COX-2, iNOS, and MMP-9 levels, indicating the involvement of DNA damage and inflammation in CEES-caused skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-caused DNA damage and the induction of inflammatory molecules. Oral GSH (300mg/kg) administration 1 h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injuries involve DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injuries in humans by SM.

Acrylamide (ACR) intoxication is associated with selective nerve terminal damage in the central and peripheral nervous systems. As a soft electrophile, ACR could form adducts with nucleophilic sulfhydryl groups on cysteine residues of kelch-like erythroid cell-derived protein with CNS homology-associated protein 1 (Keap1) leading to dissociation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activation of the antioxidant-responsive element (ARE) and subsequent upregulated gene expression of phase II detoxification enzymes and anitoxidant proteins might provide protection in neuronal regions with transcriptional capabilities (e.g., cell body). In contrast, non-transcriptional cell regions (axons, nerve terminals) might be vulnerable to electrophile-induced damage. To test this possibility, immunoblot analysis was used to measure protein products of Nrf2-activated ARE genes in nerve terminals and in cytosolic/nuclear factions of neuronal cell bodies isolated from rats intoxicated at two different ACR dose-rates; i.e., 50 mg/kg/d × 10 days, 21 mg/kg/d × 38 days. To detect possible differences in cell-specific induction, the cytoprotective response to ACR intoxication was determined in hepatic cells. Results show that control brain and hepatic cell fractions exhibited distinct subcellular distributions of Nrf2, Keap1 and several ARE protein products. ACR intoxication, however, did not alter the levels of these proteins in synaptosomal, brain cytoplasm or liver cell fractions. These data indicate that ACR was an insufficient electrophilic signal for ARE induction in all subcellular fractions tested. Because a cytoprotective response was not induced in any fraction, nerve terminal vulnerability to ACR cannot be ascribed to the absence of transcription-based defense mechanisms in this neuronal region.

PCBs, a group of 209 individual congeners, are ubiquitous environmental pollutants and classified as probable human carcinogens. One major route of exposure is by inhalation of these industrial compounds, possibly daily from inner city air and/or indoor air in contaminated buildings. Hallmarks of aging and carcinogenesis are changes in telomere length and telomerase activity. We hypothesize that semi-volatile PCBs, like those found in inner city air, are capable of disrupting telomerase activity and altering telomere length. To explore this possibility, we exposed human skin keratinocytes to a synthetic Chicago Airborne Mixture (CAM) of PCBs, or the prominent airborne PCB congeners, PCB28 or PCB52 for up to 48 days and determined telomerase activity, telomere length, cell proliferation, and cell cycle distribution. PCBs 28, 52 and CAM significantly reduced telomerase activity from days 18–48. Telomere length was shortened by PCB52 from day 18 and PCB28 and CAM from days 30 on. All PCBs decreased cell proliferation from day 18; only PCB52 produced a small increase of cells in G0/G1 of the cell cycle. This significant inhibition of telomerase activity and reduction of telomere length by PCB congeners suggest a potential mechanism by which these compounds could lead to accelerated aging and cancer.

Recent studies indicate that there is interaction between the glutamatergic neurotransmitters system and lead neurotoxicity. Previously, we have demonstrated the potential effects of glutamate in lead-induced cell death in PC12 cells and the protective role of the novel thiol antioxidant, N-acetylcysteine amide (NACA). The current study 1) investigated the potential effects of glutamate on lead exposed CD-1 mice and 2) evaluated the protective effects of NACA against glutamate and lead toxicity in CD-1 mice, and 3) compared the results with N-aceytylcysteine (a well-known thiol antioxidant). Oxidative stress parameters, including glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, and malondialdehyde (MDA) levels, were evaluated. Blood and tissue lead levels, glutamate/glutamine (Glu/Gln) ratios, GS activity, and phospholipase-A2 (PLA2) were also analyzed. Results indicated that lead and glutamate decreased GSH levels in the red blood cells, brains, livers, and kidneys. Exposure to glutamate and lead elevated the MDA levels and PLA2 activity. NACA and NAC provided protection against the detrimental effects of lead by decreasing the blood and tissue lead levels, restoring intracellular GSH levels, and decreasing the MDA levels. NACA and NAC also increased the GS activity thereby decreasing Glu/Gln levels. However, NACA appeared to have better chelating and antioxidant properties than NAC, due to its higher liphophilicity and its ability to cross the blood-brain barrier.

In human liver, the two-electron reduction of quinone compounds such as menadione is catalyzed by cytosolic carbonyl reductase (CBR) and NAD(P)H:quinone oxidoreductase (NQO1) activities. We assessed the relative contributions of CBR and NQO1 activities to the total menadione reducing capacity in liver cytosols from black (n = 31) and white donors (n = 63). Maximal menadione reductase activities did not differ between black (13.0 ± 5.0 nmol/min.mg), and white donors (11.4 ± 6.6 nmol/min.mg; p = 0.208). In addition, both groups presented similar levels of CBR activities (CBRblacks = 10.9 ± 4.1 nmol/min.mg versus CBRwhites = 10.5 ± 5.8 nmol/min.mg; p = 0.708). In contrast, blacks showed higher NQO1 activities (two-fold) than whites (NQO1blacks = 2.1 ± 3.0 nmol/min.mg versus NQO1whites =0.9 ± 1.6 nmol/min.mg, p < 0.01). To further explore this disparity, we tested whether NQO1 activity was associated with the common NQO1*2 genetic polymorphism by using paired DNA samples for genotyping. Cytosolic NQO1 activities differed significantly by NQO1 genotype status in whites (NQO1whites[NQO1*1/*1] = 1.3 ± 1.7 nmol/min.mg versus NQO1whites[NQO1*1/*2 + NQO1*2/*2] = 0.5 ± 0.7 nmol/min.mg, p < 0.01), but not in blacks (NQO1blacks[NQO1*1/*1] = 2.6 ± 3.4 nmol/min.mg versus NQO1blacks[NQO1*1/*2] = 1.1 ± 1.2 nmol/min.mg, p = 0.134). Our findings pinpoint the presence of significant interethnic differences in polymorphic hepatic NQO1 activity.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate xenobiotic sensing and metabolism through interactions with multiple exogenous and endogenous chemicals. Compounds that activate CAR are often ligands of PXR; attention is therefore given to discovery of new, receptor-specific chemical entities that may be exploited for therapeutic and basic research purposes. Recently, ligands of the peripheral benzodiazepine receptor (PBR), PK11195 and FGIN-1-27, were shown to modulate both CAR and PXR. PBR is a mitochondrial transport protein responsible for multiple regulatory functions, including heme biosynthesis, a major component in cytochrome P450 (CYP) enzymes. To investigate possible new roles for PBR involvement in metabolic regulation, expression of the CAR and PXR target genes, CYP2B6 and CYP3A4, was measured in human hepatocytes following treatment with a targeted PBR ligand set. Luciferase reporter assays with transiently expressed wild-type CAR (CAR1), splice variant CAR3, or PXR in HuH-7 cells were used to further study activation of these receptors. Four structurally-related PBR ligands (benzothiazepines) differentially modulate CAR1, CAR3 and PXR activity. Benzothiazepine NF49 is an agonist ligand of CAR3, a partial agonist of PXR, exhibits greater inverse agonist activity on CAR1 than does PK11195, and is a new tool for studying these closely related nuclear receptors.

Environmental factors (e.g., BaP) have been pointed out as one of the etiologies of pancreatic cancer. However, very limited experimental assays are available to identify pancreatic specific environmental mutagens or susceptibility genes. In this study, we have developed a simple in vitro cell culture model system that can be used to study the molecular and biochemical aspects of carcinogenesis in a near-normal immortalized pancreatic ductal epithelial cell lines. In order to demonstrate that xenobiotic stress response is intact in these cells we employed standard molecular biology techniques. For examples, luciferase reporter and/or real-time quantitative PCR assays were used to determine stress-induced CYP1A1 and CYP1B1 gene expression. Western blotting and immunocytochemistry assays were used to demonstrate that TCDD or BaP could activate AhR signaling. For exploring the carcinogenesis mechanism, we incubated cells with [3H]BaP and determined BaP-DNA binding activity by measuring its radioactivity. BaP-DNA adduct formation was further confirmed by [32P]-postlabeling assay. Finally, we demonstrated the effects of endogenous AhR or BRCA1 in BaP-DNA adduct accumulation in our cell system: As results, no apparent BaP-DNA adduct accumulation by [32P]-postlabeling assay was found in either control-siRNA or AhR-siRNA pretreated cells. On the other hand, a significant increase of BaP-DNA adduct accumulation was found in BRCA1 knockdown cells. In conclusion, we suggest that this in vitro model may provide the feasibility for future studies on the molecular basis of pancreatic ductal cell carcinogenesis caused by dietary mutagens.

Cigarette smoking is a devastating risk factor for cardiovascular diseases and nicotine is believed the main toxin component responsible for the toxic myocardial effects of smoking. Nonetheless, neither the precise mechanism of nicotine -induced cardiac dysfunction nor effective treatment is elucidated. The aim of this study was to evaluate the impact of cardiac-specific overexpression of heavy metal scavenger metallothionein on myocardial geometry and mechanical function following nicotine exposure. Adult male FVB wild-type and metallothionein mice were injected with nicotine (2 mg/kg/d) intraperitoneally for 10 days. Mechanical and intracellular Ca2+ properties were examined. Myocardial histology (cross-sectional area and fibrosis) was evaluated by H&E and Masson trichrome staining, respectively. Oxidative stress and apoptosis were measured by CM-H2DCFDA fluorescence and caspase-3 activity, respectively. Nicotine exposure failed to affect the protein abundance of metallothionein. Our data revealed reduced echocardiographic contractile capacity (fractional shortening), altered cardiomyocyte contractile and intracellular Ca2+ properties including depressed peak shortening amplitude, maximal velocity of shortening/ relengthening, resting and electrically-stimulated rise in intracellular Ca2+, as well as prolonged duration of relengthening and intracellular Ca2+ clearance in hearts from nicotine-treated FVB mice, the effect of which was ameliorated by metallothionein. Biochemical and histological findings depicted overt accumulation of ROS, apoptosis and myocardial fibrosis without any change in myocardial cross-sectional area following nicotine treatment, which was mitigated by metallothionein. Taken together, our findings suggest the antioxidant metallothionein may reconcile short-term nicotine exposure-induced myocardial contractile dysfunction and fibrosis possibly through inhibition of ROS accumulation and apoptosis.