Mary Beth Genter

Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is a pervasive environmental contaminant that induces hepatic and extrahepatic oxidative stress. We have previously shown that dioxin increases mitochondrial respiration-dependent reactive oxygen production. In the present study we examined the dependence of mitochondrial reactive oxygen production on the aromatic hydrocarbon receptor (AHR), cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1A2 (CYP1A2), proteins believed to be important in dioxin-induced liver toxicity. Congenic Ahr(-/-), Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and C57BL/6J inbred mice as their Ahr/Cyp1a1/Cyp1a2(+/+) wild-type (wt) counterparts, were injected intraperitoneally with dioxin (15 microg/kg body weight) or corn-oil vehicle on 3 consecutive days. Liver mitochondria were examined 1 week following the first treatment. The level of mitochondrial H(2)O(2) production in vehicle-treated Ahr(-/-) mice was one fifth that found in vehicle-treated wt mice. Whereas dioxin caused a rise in succinate-stimulated mitochondrial H(2)O(2) production in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, this increase did not occur with the Ahr(-/-) knockout. The lack of H(2)O(2) production in Ahr(-/-) mice was not due to low levels of Mn(2+)-superoxide dismutase (SOD2) as shown by Western immunoblot analysis, nor was it due to high levels of mitochondrial glutathione peroxidase (GPX1) activity. Dioxin decreased mitochondrial aconitase (an enzyme inactivated by superoxide) by 44% in wt mice, by 26% in Cyp1a2(-/-) mice, and by 24% in Cyp1a1(-/-) mice; no change was observed in Ahr(-/-) mice. Dioxin treatment increased mitochondrial glutathione levels in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, but not in Ahr(-/-) mice. These results suggest that both constitutive and dioxin-induced mitochondrial reactive oxygen production is associated with a function of the AHR, and these effects are independent of either CYP1A1 or CYP1A2.

Olfactory toxicity of methimazole: dose-response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa.

Methimazole is a compound administered to humans for the treatment of hyperthyroidism and is used experimentally as a model substrate for the flavin-containing monooxygenase (FMO) system. Previous results from this laboratory demonstrated that methimazole is an olfactory system toxicant, causing nearly complete destruction of the olfactory epithelium in the male Long-Evans rat following a single ip dose of 300 mg/kg. The present studies were undertaken to determine the dose-response relationship for methimazole-induced olfactory mucosal damage and to determine whether or not similar damage occurs as a result of oral administration, mimicking the relevant route of human exposure. We also investigated the mechanism of olfactory toxicity of methimazole by means of a structure-activity study and began the characterization of the form(s) of FMO present in the olfactory mucosa of the male Long-Evans rat. Dose-response analysis demonstrated that methimazole causes olfactory mucosal damage at doses of 25 mg/kg ip and greater. The results of gavage studies showed that a single oral dose of 50 mg/kg also caused olfactory mucosal damage. Two structurally related compounds, methylimidazole and methylpyrrole, were not olfactory toxicants, suggesting that a reactive intermediate generated in the course of metabolizing methimazole to an S-oxide is the olfactory toxic species. Microsomal incubation studies revealed the presence ofmethimazole S-oxidation activity in olfactory mucosal microsomes at levels comparable to those in liver. An anti-mouse liver FMO antibody reacted on Western blots with olfactory mucosal microsomes. These findings demonstrate a dose-response for the olfactory toxicity of methimazole and suggest that characterization of human olfactory mucosal FMO activity may be necessary to assess the potential for human risk associated with therapeutic exposure to methimazole.

Evidence that pyrrole formation is a pathogenetic step in γ-diketone neuropathy☆

Abstract Previous studies from this laboratory have demonstrated that the addition of methyl groups at the 3 and 4 positions of the 2,5-hexanedione (2,5-HD) molecule results both in more rapid pyrrole formation and in enhanced neurotoxicity. In order to define more clearly the relationship between rates of pyrrole formation and neurotoxicity, the dl and meso diastereomers of 3,4-dimethyl-2,5-hexanedione (DMHD), 3,4-diethyl-2,5-hexanedione (DEHD), and 3,4-diisopropyl-2,5-hexanedione (DiPHD) were synthesized and purified. The rates of pyrrole formation were compared with that of unsubstituted 2,5-HD, and rates of in vitro crosslinking were determined. Each of the compounds was administered to rats to determine relative neurotoxicity. Hindlimb paralysis was reached after a total administered dose of 1.6 mmol/kg of dl -DMHD, while 5.9 mmol/kg of meso -DMHD was required. Paralysis was not achieved with either diastereomer of DEHD or DiPHD, although both produced systemic toxicity. Histologic sections of spinal cords and anterior roots from rats treated with DMHD revealed large neurofilament-filled axonal swellings, while more distal sections contained axons undergoing Wallerian-type degeneration. Neither axonal swellings nor Wallerian-type degeneration were seen in sections from spinal cord or peripheral nerve of rats treated with DEHD or DiPHD. The rates of pyrrole formation were in the order dl -DMHD > meso -DMHD > 2,5-HD > dl -DEHD > meso -DEHD > dl -DiPHD . meso -DiPHD, while in vitro crosslinking rates were in the order dl -DMHD > meso -DMHD . dl -DEHD > meso -DEHD > 2,5-HD > dl -DiPHD > meso -DiPHD. Cyclic voltammetry showed that the autoxidation of pyrroles derived from DMHD, DEHD, and DiPHD occurred more readily than that derived from 2,5-HD. In addition, we report for the first time the segregation of axoplasmic organelles in animals treated with DMHD, providing further evidence that the neurofilamentous axonopathies caused by such compounds as β,β′-iminodipropionitrile (IDPN), 2,5-HD and CS 2 share a common underlying mechanism. The strong correlations between rates of pyrrole formation, rates of in vitro crosslinking and relative neurotoxicity are seen as evidence that pyrrole formation is a step in the pathogenetic sequence of γ-diketone neuropathy.

Dietary Whey Protein Lowers the Risk for Metabolic Disease in Mice Fed a High-Fat Diet

Consuming a high-fat (HF) diet produces excessive weight gain, adiposity, and metabolic complications associated with risk for developing type 2 diabetes and fatty liver disease. This study evaluated the influence of whey protein isolate (WPI) on systemic energy balance and metabolic changes in mice fed a HF diet. Female C57BL/6J mice received for 11 wk a HF diet, with or without 100 g WPI/L drinking water. Energy consumption and glucose and lipid metabolism were examined. WPI mice had lower rates of body weight gain and percent body fat and greater lean body mass, although energy consumption was unchanged. These results were consistent with WPI mice having higher basal metabolic rates, respiratory quotients, and hepatic mitochondrial respiration. Health implications for WPI were reflected in early biomarkers for fatty liver disease and type 2 diabetes. Livers from WPI mice had significantly fewer hepatic lipid droplet numbers and less deposition of nonpolar lipids. Furthermore, WPI improved glucose tolerance and insulin sensitivity. We conclude that in mice receiving a HF diet, consumption of WPI results in higher basal metabolic rates and altered metabolism of dietary lipids. Because WPI mice had less hepatosteatosis and insulin resistance, WPI dietary supplements may be effective in slowing the development of fatty liver disease and type 2 diabetes.

Distribution and Systemic Effects of Intranasally Administered 25 nm Silver Nanoparticles in Adult Mice

Previous work indicates that silver nanoparticles (AgNPs) given IP to mice alter the regulation of inflammation- and oxidative stress–related genes in brain. Here we assessed the distribution and toxic potential of AgNP following intranasal (IN) exposure. Adult male C57BL/6J mice received 25-nm AgNP (100 or 500 mg/kg) once IN. After 1 or 7 days, histopathology of selected organs was performed, and tissue reduced glutathione (GSH) levels were measured as an indicator of oxidative stress. Aggregated AgNP were found in spleen, lung, kidney, and nasal airway by routine light microscopy. Splenic AgNP accumulation was greatest in red pulp and occurred with modestly reduced cellularity and elevated hemosiderin deposition. Aggregated AgNP were not associated with microscopic changes in other tissues except for nasal mucosal erosions. Autometallography revealed AgNP in olfactory bulb and the lateral brain ventricles. Neither inflammatory cell infiltrates nor activated microglia were detected in brains of AgNP-treated mice. Elevated tissue GSH levels was observed in nasal epithelia (both doses at 1 day, 500 mg/kg at 7 days) and blood (500 mg/kg at 7 days). Therefore, IN administration of AgNP permits systemic distribution, produces reversible oxidative stress in the nose and in blood, and mildly enhances macrophage-mediated erythrocyte destruction in the spleen.

Lipid metabolism and body composition in Gclm(-/-) mice

In humans and experimental animals, high fat diets (HFD) are associated with risk factors for metabolic diseases, such as excessive weight gain and adiposity, insulin resistance and fatty liver. Mice lacking the glutamate-cysteine ligase modifier subunit gene (Gclm(-/-)) and deficient in glutathione (GSH), are resistant to HFD-mediated weight gain. Herein, we evaluated Gclm-associated regulation of energy metabolism, oxidative stress, and glucose and lipid homeostasis. C57BL/6J Gclm(-/-) mice and littermate wild-type (WT) controls received a normal diet or an HFD for 11 weeks. HFD-fed Gclm(-/-) mice did not display a decreased respiratory quotient, suggesting that they are unable to process lipid for metabolism. Although dietary energy consumption and intestinal lipid absorption were unchanged in Gclm(-/-) mice, feeding these mice an HFD did not produce excess body weight nor fat storage. Gclm(-/-) mice displayed higher basal metabolic rates resulting from higher activities of liver mitochondrial NADH-CoQ oxidoreductase, thus elevating respiration. Although Gclm(-/-) mice exhibited strong systemic and hepatic oxidative stress responses, HFD did not promote glucose intolerance or insulin resistance. Furthermore, HFD-fed Gclm(-/-) mice did not develop fatty liver, likely resulting from very low expression levels of genes encoding lipid metabolizing enzymes. We conclude that Gclm is involved in the regulation of basal metabolic rate and the metabolism of dietary lipid. Although Gclm(-/-) mice display a strong oxidative stress response, they are protected from HFD-induced excessive weight gain and adipose deposition, insulin resistance and steatosis.

In Utero and Lactational Exposure to PCBs in Mice: Adult Offspring Show Altered Learning and Memory Depending on CYP1a2 and AhR Genotypes

Background: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2)-uninduced basal levels and > 12-fold variability in aryl hydrocarbon receptor (AHR)affinity; because CYP1A2 is known to sequester coplanar PCBs and because AHR ligands include coplanar PCBs, both genotypes can affect PCB response. Objectives: We aimed to develop a mouse paradigm with extremes in Cyp1a2 and Ahr genotypes to explore genetic susceptibility to PCB-induced developmental neurotoxicity using an environmentally relevant mixture of PCBs. Methods: We developed a mixture of eight PCBs to simulate human exposures based on their reported concentrations in human tissue, breast milk, and food supply. We previously characterized specific differences in PCB congener pharmacokinetics and toxicity, comparing high-affinity–AHR Cyp1a2 wild-type [Ahrb1_Cyp1a2(+/+)], poor-affinity–AHR Cyp1a2 wild-type [Ahrd_Cyp1a2(+/+)], and high-affinity–AHR Cyp1a2 knockout [Ahrb1_Cyp1a2(–/–)] mouse lines [Curran CP, Vorhees CV, Williams MT, Genter MB, Miller ML, Nebert DW. 2011. In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes. Toxicol Sci 119:189–208]. Dams received a mixture of three coplanar and five noncoplanar PCBs on gestational day 10.5 and postnatal day (PND) 5. In the present study we conducted behavioral phenotyping of exposed offspring at PND60, examining multiple measures of learning, memory, and other behaviors. Results: We observed the most significant deficits in response to PCB treatment in Ahrb1_Cyp1a2(–/–) mice, including impaired novel object recognition and increased failure rate in the Morris water maze. However, all PCB-treated genotypes showed significant differences on at least one measure of learning or behavior. Conclusions: High levels of maternal hepatic CYP1A2 offer the most important protection against deficits in learning and memory in offspring exposed to a mixture of coplanar and noncoplanar PCBs. High-affinity AHR is the next most important factor in protection of offspring.

Uptake of materials from the nasal cavity into the blood and brain: are we finally beginning to understand these processes at the molecular level?

Substances that enter the nasal cavity can access the bloodstream or central nervous system by processes including receptor cell uptake, transneuronal transport, and paracellular transport. Until recently, the molecular mechanisms by which agents move from the nasal cavity have not been described. Although the full complement of transporter proteins found in the nasal cavity has certainly not yet been identified, several recent observations have advanced this field substantially. We summarize here a representative sample of transporter proteins found in olfactory mucosa and/or nasal respiratory mucosa and the substrates that they transport into the brain and/or bloodstream.

Acetaminophen normalizes glucose homeostasis in mouse models for diabetes.

Loss of pancreatic beta cell insulin secretion is the most important element in the progression of type 1 and type 2 diabetes. Since oxidative stress is involved in the progressive loss of beta cell function, we evaluated the potential for the over-the-counter analgesic drug and antioxidant, acetaminophen (APAP), to intervene in the diabetogenic process. We used mouse models for type 1 diabetes (streptozotocin) and type 2 diabetes (high-fat diet) to examine the ability of APAP to intervene in the progression of diabetes. In C57BL/6J mice, streptozotocin caused a dosage dependent increase in fasting blood glucose (FBG), from 100 to >600mg/dl. Daily APAP (20mg/kg BW, gastric gavage), significantly prevented and partially reversed the increase in FBG levels produced by streptozotocin. After 10 weeks on a high-fat diet, mice developed fasting hyperinsulemia and impaired glucose tolerance compared to animals fed a control diet. APAP largely prevented these changes in insulin and glucose tolerance. Furthermore, APAP prevented most of the increase in body fat in mice fed the high-fat diet. One protective mechanism for APAP is suggested by studies using isolated liver mitochondria, where low micromolar concentrations abolished the production of reactive oxygen that might otherwise contribute to the destruction of pancreatic beta-cells. These findings suggest that administration of APAP to mice, in a dosage used safely by humans, reduces the production of mitochondrial reactive oxygen and concomitantly prevents the development of type 1 and type 2 diabetes in established animal models.

Manganese accumulation in the mouse ear following systemic exposure.

There is evidence in human populations that exposure to manganese (Mn), or Mn in combination with excessive noise exposure, results in hearing loss. Quantitative reverse‐transcriptase polymerase chain reaction revealed expression of the metal transporters DMT1, ZIP8, and ZIP14 in control mouse ears. ZIP8 is known to have a high affinity (Km = 2.2 µM) for Mn transport, and ZIP8 protein was localized to the blood vessels of the ear by immunohistochemistry. We treated mice (strains C57BL/6J and DBA/2J) with Mn (100 mg/kg MnCl2, by subcutaneous injection, on three alternating days), and Mn was significantly elevated in the ears of the treated mice. Mn concentrations remained elevated over controls for at least 2 weeks after treatment. These studies demonstrate that metal transporters are present in the mouse ear and that Mn can accumulate in the ear following systemic exposure. Future studies should focus on whether Mn exposure is associated with hearing deficits.