Modulation of glutathione and glutamate-L-cysteine ligase by methylmercury during mouse development.

The antioxidant tripeptide glutathione has been proposed to be important in defense against oxidative stress and heavy metal toxicity. We evaluated alterations in glutathione regulation and synthesis associated with low-level chronic methylmercury (MeHg) exposure in the developing mouse fetus. Female C57Bl/6 mice were given 0, 3, or 10 ppm MeHg in the drinking water for 2 weeks prior to breeding and throughout pregnancy. Fetuses were collected on gestational days (gd) 12 and 16. Total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSR), and glutamate-L-cysteine ligase (Glcl) activity were assessed in yolk sacs and fetuses at gd 16. Western and Northern blots for Glcl-catalytic (Glclc) and Glcl-regulatory (Glclr) subunits were performed on gd 12 and gd 16 fetuses. There were no changes in total glutathione in gd 16 mouse fetuses with exposure, but there were dose-related decreases in GSH and increases in GSSR. In contrast, visceral yolk sacs exhibited an increase in total glutathione in the low-dose groups, but no changes in the high-dose group. There were no changes in Glcl activity in fetuses, but there was a 2-fold increase in Glcl activity in yolk sacs from both low-dose and high-dose groups. There was a 2-fold induction in GLCLC: mRNA and protein in the gd 16 yolk sacs at both 3 and 10 ppm MeHg. No treatment-related changes in Glclr protein in either gd 12 or gd 16 yolk sacs or fetuses were found. Thus, the yolk sac is capable of up-regulating Glclc and GSH synthetic capacity in response to MeHg exposure. This increase appears to be sufficient to resist MeHg-induced GSH depletion in the yolk sac; however fetal glutathione redox status is compromised with exposure to 10 ppm MeHg.     

Repeated inhalation exposures to the bioactivated cytotoxicant naphthalene (NA) produce airway-specific Clara cell tolerance in mice.

Repeated exposures to bioactivated cytotoxicants such as naphthalene (NA) render the target population, Clara cells, resistant to further injury through a glutathione-dependent mechanism. The current studies were designed to test the hypothesis that the mechanism for tolerance is localized in Clara cells. We used three approaches to test this hypothesis. First, using airway explants from tolerant mice maintained in culture, we sought to determine if the mechanism of Clara cell tolerance was airway-specific. Second, using inhalation as the route of exposure, we sought to determine if Clara cells at all airways levels become tolerant to repeated inhalation exposures of NA. Third, by measuring gamma-glutamylcysteine synthetase (gamma-GCS) activity and expression we determined if tolerance to inhaled NA resulted from shifts in phase-II metabolism. Our results indicate that Clara cells in explants from tolerant mice remained tolerant to NA injury in culture. When mice were exposed to repeated inhalation exposures of NA (15 ppm), we found that Clara cells at all airway levels became tolerant. Expression and activity analysis revealed that gamma-GCS, the rate-limiting enzyme in glutathione synthesis, is induced in tolerant Clara cells. Buthionine sulfoximine, a gamma-GCS inhibitor, was able to eliminate the resistance of these tolerant cells. We conclude: (1) the mechanism of NA tolerance in Clara cells is airway specific, (2) the specific mechanism allows Clara cells to become tolerant to NA vapor at levels relevant to human exposure, and (3) the mechanism of tolerance to inhaled NA is highly dependent on induction of the catalytic enzyme, gamma-GCS.     

15-deoxy prostaglandin J2 enhances allyl alcohol-induced toxicity in rat hepatocytes.

Allyl alcohol causes hepatotoxicity that is potentiated by small doses of bacterial lipopolysaccharide (LPS) through a cyclooxygenase-2 (COX-2)-dependent mechanism. The COX-2 product prostaglandin D(2) (PGD(2)) increases hepatocyte killing by allyl alcohol in vitro. In the present study the ability of the nonenzymatic product of PGD(2), 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), to increase the cytotoxicity of allyl alcohol was evaluated. In a concentration-dependent manner, 15d-PGJ(2) significantly augmented cell death caused by allyl alcohol in isolated rat hepatocytes. 15d-PGJ(2) also increased the cytotoxicity of acrolein, the active metabolite of allyl alcohol. An agonist for the PGD(2) receptor neither reproduced the increase in allyl alcohol-mediated cytotoxicity nor altered the response to 15d-PGJ(2). Similarly, these responses were not affected by either an agonist or an antagonist for the peroxisome proliferator-activated receptor-gamma. The enhancement by 15d-PGJ(2) of allyl alcohol-mediated cell killing was unaffected by augmentation or inhibition of cAMP. Protein synthesis was markedly decreased by 15d-PGJ(2), but inhibition of protein synthesis alone with cycloheximide did not increase allyl alcohol-mediated cell killing. Allyl alcohol at subtoxic concentrations increased translocation of nuclear factor kappa B (NF-kappaB), whereas at cytotoxic concentrations no translocation occurred. 15d-PGJ(2) inhibited translocation of NF-kappaB from the cytosol to the nucleus both in the presence and absence of allyl alcohol. Like 15d-PGJ(2), MG132, an inhibitor of NF-kappaB activation, enhanced allyl alcohol-induced hepatocyte death. Together these results indicate that 15d-PGJ(2) augments hepatocyte killing by allyl alcohol, and the mechanism may be related to the inhibition of NF-kappaB activation.     

Nonclinical safety evaluation of muraglitazar, a novel PPARalpha/gamma agonist.

The toxicity of muraglitazar, an oxybenzylglycine, nonthiazolidinedione peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, was evaluated in a comprehensive nonclinical toxicology program that included single-dose oral toxicity studies in mice, rats, and monkeys; repeat-dose toxicity studies in rats, dogs, and monkeys; a battery of in vitro and in vivo genetic toxicity studies; carcinogenicity studies in mice and rats; reproductive and developmental toxicity studies in rats and rabbits; and studies to investigate species-specific findings. Pharmacologically mediated changes, similar to those observed with other PPARgamma agonists, were observed following chronic administration and included subcutaneous edema, hematologic/hematopoietic and serum chemistry alterations, and morphologic findings in the heart and adipose tissue in rats and monkeys. In dogs, a species highly sensitive to PPARgamma agonists, muraglitazar caused pronounced species-specific clinical toxicity and degenerative changes in the brain, spinal cord, and testes at high doses and exposures. Muraglitazar was nongenotoxic in the standard battery of genotoxicity studies. Gallbladder adenomas in male mice and adipocyte neoplasms in male and female rats were seen at suprapharmacologic exposures, whereas urinary bladder tumors occurred in male rats at lower exposures. Subsequent investigative studies established that the urinary bladder carcinogenic effect was mediated by urolithiasis rather than a direct pharmacologic effect on urothelium. Muraglitazar had no effects on reproductive function in male and female rats at high systemic exposures, was not teratogenic in rats or rabbits, and demonstrated no selective developmental toxicity. Overall, there were no nonclinical findings that precluded the safe administration of muraglitazar to humans.     

Induction of chemokines by low-dose intratracheal silica is reduced in TNFR I (p55) null mice.

Previous studies suggest that tumor necrosis factor alpha (TNF-alpha) and the TNFRI (p55) and TNFRRII (p75) receptors mediate the pulmonary fibrotic response to silica. In order to further define the role of the TNFRI (p55) receptor in induction of profibrotic chemokines by low-dose silica/crystalline silica (50 micro g/50 micro l/mouse) or control diluent saline was instilled into the trachea of TNFRI gene ablated ((-/-)) and C57BL/6 (WT) control mice. Lung tissue was harvested and bronchoalveolar lavage (BAL) performed 24 h and 28 days following silica administration. Selected profibrotic chemokine mRNAs were quantified by ribonuclease protection assay, normalized to ribosomal protein L32 mRNA content and expressed relative to saline control treated lungs. Induction of MIP-1beta, MIP-1alpha, MIP-2, IP-10, and MCP-1 mRNAs was attenuated in the TNFRI(-/-) mice, in comparison to WT mice, particularly at 28 days after exposure. ELISA assays for MIP-1alpha and MIP-2 in homogenized lung tissue similarly demonstrated marked induction of both chemokines 24 h after silica treatment, which was persistent at 28 days in WT but not in TNFRI(-/-) mice. The percentage of BAL cells that was neutrophils was comparably increased in WT and RI(-/-) lungs at 24 h (49 +/- 12% vs. 46 +/- 10%) and 28 days (6.2 +/- 1.5% vs. 4.5 +/- 1%). The increase in total lavagable cells and BAL protein was also independent of strain. Histology revealed mild alveolitis without granuloma formation in both strains, slightly decreased in TNFRI(-/-). This study demonstrates an increase in pro-fibrotic chemokines in response to a single intratracheal exposure to crystalline silica that was sustained at 28 days after treatment in WT but not in TNFRI(-/-) mice. Silica dependent recruitment of neutrophils to the alveolar space and alveolar protein leak were, however, not altered by the absence of the TNF receptor.     

Divergent roles for glutathione in lindane-induced acute and delayed-onset inhibition of rat myometrial gap junctions.

Previous studies have shown that the insecticide lindane (gamma-hexachlorocyclohexane) induces a biphasic inhibition of gap junction intercellular communication that is accompanied by oxidative stress. The present study investigates the hypothesis that depletion of cellular glutathione (GSH) is a mechanistic link between lindane-induced oxidative stress and inhibition of myometrial gap junctions. Exposure to 100 or 200 microM lindane rapidly (within 1 min) increased myometrial cell generation of superoxide, as measured by superoxide dismutase-inhibitable cytochrome c reduction, and superoxide production remained elevated for up to 60 min of exposure. To measure gap junction communication, Lucifer yellow dye was injected into myometrial cells, and dye transfer to adjoining cells was monitored. Cells were exposed to lindane with or without GSH modulators, and dye transfer was determined at the end of a 1-h exposure to 100 microM lindane (acute phase) and 24 h after termination of lindane exposure (secondary phase). The acute phase of lindane-induced inhibition of dye transfer was prevented by GSH depletion with L-buthionine-[S,R]-sulfoximine (BSO) and enhanced by GSH augmentation with GSH monoethyl ester or L-2-oxothiazolidine-4-carboxylate (OTC). In contrast, the secondary, delayed-onset phase of lindane-induced inhibition of dye transfer was enhanced by GSH depletion with BSO and prevented by GSH augmentation with GSH monoethyl ester or OTC. Changes in cellular GSH by the pharmacological modulators were confirmed by high performance liquid chromatography. These results suggest that GSH is required in the acute phase but protects against the secondary phase of lindane-induced inhibition of myometrial gap junctions.     

Role of lipid peroxidation as a mechanism of liver injury after acetaminophen overdose in mice.

Mitochondrial oxidant stress and peroxynitrite formation have been implicated in the pathophysiology of acetaminophen-induced (AAP-induced) liver injury. Therefore, we tested the hypothesis that lipid peroxidation (LPO) might be involved in the injury mechanism. Male C3Heb/FeJ mice fed a diet high in vitamin E (1 g d-alpha-tocopheryl acetate/kg diet) for 1 week had 6.7-fold higher hepatic tocopherol levels than animals on the control diet (8.2 +/- 0.1 nmol/g liver). Treatment of fasted mice with 300 mg/kg AAP caused centrilobular necrosis with high plasma alanine aminotransferase (ALT) activities at 6 h (3280 +/- 570 U/l) but no evidence of LPO (hepatic malondialdehyde, 4-hydroxynonenal). Animals on the vitamin E diet had similar injury and LPO as mice on the control diet. To verify a potential effect of the vitamin E diet on drug-induced liver injury, animals were pretreated with a combination of phorone, FeSO4, and allyl alcohol. We observed, 2 h after allyl alcohol, massive LPO and liver cell injury in the livers of animals on the control diet, as indicated by a 32-fold increase in malondialdehyde levels, extensive staining for 4-hydroxynonenal, and ALT activities of 2310 +/- 340 U/l. Animals on the vitamin E diet had 40% lower hepatic malondialdehyde levels and 85% lower ALT values. Similar results were obtained when animals were treated for 3 days with alpha- or gamma-tocopherol (0.19 mmol/kg, ip). Both treatments reduced LPO and injury after allyl alcohol but had no effect on AAP hepatotoxicity. Thus, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation, LPO does not appear to be a critical event in AAP-induced hepatotoxicity.     

An L-tyrosine derivative and PPARgamma agonist, GW7845, activates a multifaceted caspase cascade in bone marrow B cells.

Apoptosis is a critical event in the deletion of B lymphocytes prior to their migration to the periphery. Synthetic peroxisome proliferator activated receptor gamma (PPARgamma) agonists, including the drug GW7845 and the environmental contaminant mono-(2-ethylhexyl) phthalate, as well as an endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), induce clonally unrestricted apoptosis in pro/pre-B cells. Considering that PPARgamma agonists are used clinically for the treatment of diabetes and postulated to be useful as chemotherapeutics, we used GW7845 as a model PPARgamma agonist to examine the mechanism of cell death that may contribute to tumor killing as well as normal bone marrow B lymphocyte toxicity. GW7845 induced rapid mitochondrial membrane depolarization and release of cytochrome c, along with nearly concurrent activation of capases-2, -3, -8, and -9 in primary pro-B cells and BU-11 cells, a nontransformed pro/pre-B cell line. GW7845-induced apoptosis was reduced significantly in Bax-deficient and Apaf-1 mutant primary pro-B cells, supporting the conclusion that GW7845-induced apoptosis is mitochondria- and apoptosome-dependent. Using benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK) as a pan-caspase inhibitor, we demonstrated that an initial cytochrome c release occurred independently of caspase activation and that only caspase-9 activation was partially caspase independent. The attenuation of GW7845-induced apoptosis by multiple FMK-labeled peptide sequences suggests that multiple caspase pathways are responsible for initiating and executing apoptosis. The strong activation of Bid provides a mechanism by which caspases-2, -3, and -8 may amplify the apoptotic signal. These data support the hypothesis that pharmacologic concentrations of PPARgamma agonists induce an intrinsic apoptotic pathway that is driven in normal bone marrow B cells by multiple amplification loops.     

alpha 2u-Globulin nephropathy, renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-344 rats.

tert-Butyl alcohol (TBA) has been shown to cause kidney tumors in male rats following chronic administration in drinking water. The objective of the present study was to determine whether TBA induces alpha 2u-globulin (alpha 2u) nephropathy (alpha 2u-N) and enhanced renal cell proliferation in male, but not female, F-344 rats, and whether the dosimetry of TBA to the kidney is gender specific. Male and female F-344 rats were exposed to 0, 250, 450, or 1750 ppm TBA vapors 6 h/day for 10 consecutive days to assess alpha 2u-nephropathy and renal cell proliferation and for 1 and 8 days to evaluate the dosimetry of TBA following a single and repeated exposure scenario. Protein droplet accumulation was observed in kidneys of male rats exposed to 1750 ppm TBA, with alpha 2u-globulin immunoreactivity present in these protein droplets. A statistically significant increase in alpha 2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to 1750 ppm TBA with a exposure-related increase in renal cell proliferation. Renal alpha 2u concentration was positively correlated with cell proliferation in male rat kidney. No histological lesions or increased renal cell proliferation was observed in female rats exposed to TBA compared to controls. The TBA kidney:blood ratio was higher at all concentrations and time points in male rats compared with female rats, which suggests that TBA is retained longer in male rat kidney compared with female rat kidney. Together these data suggest that TBA causes alpha 2u-N in male rats, which is responsible for the male rat-specific increase in renal cell proliferation.     

Mitochondrial thioredoxin-2 has a key role in determining tumor necrosis factor-alpha-induced reactive oxygen species generation, NF-kappaB activation, and apoptosis.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that is involved in numerous pathologies, in part through stimulation of the mitochondrial production of reactive oxygen species (ROS). Previous studies show that in addition to mitochondrial superoxide dismutase- and glutathione-dependent systems, mitochondria also contain thioredoxin-2 (Trx2), an antioxidant protein that can detoxify ROS. The purpose of this study was to determine whether Trx2 protects against oxidative damage triggered by TNF-alpha. After a 30-min treatment in HeLa cells, TNF-alpha (5-40 ng/ml) oxidized Trx2 but not cytoplasmic Trx1. Preferential, significant Trx2 oxidation occurred within 10 min of TNF-alpha treatment. Moreover, overexpression of Trx2, but not Trx1, decreased TNF-alpha-induced ROS generation, suggesting mitochondrial compartmentation of ROS production and subsequent specific detoxification by Trx2, not Trx1. Overexpression of Trx2 or the active-site mutant C93S Trx2 was used to evaluate their downstream effects following TNF-alpha stimulation. Results showed that nuclear translocation of NF-kappaB was inhibited with Trx2 overexpression but not with the dominant negative active-site mutant C93S Trx2. Moreover, when cotransfected with a NF-kappaB-luciferase reporter and then treated with TNF-alpha, NF-kappaB activity was significantly attenuated with Trx2 overexpression but not with C93S Trx2 expression. Trx2 overexpression, but not C93S Trx2, significantly inhibited TNF-alpha-induced apoptosis as measured by terminal dUTP nick-end labeling assay. These findings support the interpretation that mitochondrial-generated ROS is a principal component in TNF-alpha-induced effects and that Trx2 blocks TNF-alpha-induced ROS generation and downstream NF-kappaB activation and apoptosis.     

Biphasic lindane-induced oxidation of glutathione and inhibition of gap junctions in myometrial cells.

The insecticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells by a mechanism involving oxidative stress. We hypothesized that oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG) and subsequent S-glutathionylation provide a mechanistic link between lindane-induced oxidative stress and lindane's inhibition of myometrial gap junction communication. Gap junction communication between cultured rat myometrial myocytes was assessed by Lucifer yellow dye transfer after microinjection. A biphasic pattern was confirmed, with dye transfer nearly abolished after 1 h of exposure to 100 microM lindane followed initially by recovery after lindane removal, and then the development 4 h after termination of lindane exposure of a delayed-onset, sustained inhibition that continued for 96 h. As measured by HPLC, cellular GSH varied over a 24-h period in a biphasic fashion that paralleled lindane-induced inhibition of dye transfer, whereas GSSG levels increased in a manner inversely related to GSH. In accordance, GSH/GSSG ratios were depressed at times when GSH and dye transfer were low. Lindane substantially increased S-glutathionylation in a concentration-dependent manner, measured biochemically by GSSG reductase-stimulated release of GSH from precipitated proteins. Furthermore, treatments that promoted accumulation of GSSG (50 microM diamide and 25 microM 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]) inhibited Lucifer yellow dye transfer between myometrial cells. Findings that lindane induced GSH oxidation to GSSG with increased S-glutathionylation, together with the diamide and BCNU results, suggest that oxidation of GSH to GSSG is a component of the mechanism by which lindane inhibits myometrial gap junctions.     

p53-independent induction of rat hepatic Mdm2 following administration of phenobarbital and pregnenolone 16alpha-carbonitrile.

Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.     

DNA damage recognition in the rat zygote following chronic paternal cyclophosphamide exposure.

The detrimental effects of preconceptional paternal exposure to the alkylating anticancer agent, cyclophosphamide, include aberrant epigenetic programming, dysregulated zygotic gene activation, and abnormalities in the offspring that are transmitted to the next generation. The adverse developmental consequences of genomic instabilities transmitted via the spermatozoon emphasize the need to elucidate the mechanisms by which the early embryo recognizes DNA damage in the paternal genome. Little information exists on DNA damage detection in the zygote. We assessed the impact of paternal cyclophosphamide exposure on phosphorylated H2AX (gammaH2AX) and poly(ADP-ribose) polymerase-1(PARP-1), biomarkers of DNA damage, to determine the capacity in the rat zygote to recognize genomic damage and initiate a response to DNA lesions. An amplified biphasic gammaH2AX response was triggered in the paternal pronucleus in zygotes sired by drug-treated males; the maternal genome was not affected. PARP-1 immunoreactivity was substantially elevated in both parental genomes, coincident with the second phase of gammaH2AX induction in embryos sired by cyclophosphamide-exposed spermatozoa. Thus, paternal exposure to a DNA damaging agent rapidly activates signals implemental for DNA damage recognition in the zygote. Inefficient repair of DNA lesions may lead to persistent alterations of the histone code and chromatin integrity, resulting in aberrant embryogenesis. We propose that the response of the early embryo to disturbances in spermatozoal genomic integrity plays a vital role in determining its outcome.