Reduced Colonic Mucosal Injury in 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Poly ADP-Ribose Polymerase (TIPARP/PARP7)-Deficient Mice.

Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7-/- mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.

Effect of 25-HydroxyVitamin D Deficiency and Its Interaction with Prednisone Treatment on Musculoskeletal Health in Growing Mdx Mice.

Duchenne muscular dystrophy (DMD) results from genetic mutations of the gene encoding dystrophin, leading to muscle inflammation and degeneration that is typically treated with glucocorticoids. DMD and its treatment with glucocorticoids result in poor bone health and high risk of fractures. Insufficient levels of 25-hydroxyvitamin D (25-hydroxy D) that may contribute to weakened bone are routinely found in DMD patients. To determine the effect of 25-hydroxy D deficiency, this study examined the effects of low vitamin D dietary intake with and without glucocorticoids on the musculoskeletal system of the Mdx mouse model of DMD. At 10 weeks of age, Mdx mice on control diet had low trabecular bone mineral density of distal femurs and lumbar vertebrae with increased osteoclast numbers compared to wild-type mice. Low vitamin D intake resulted in 25-hydroxy D deficiency but had no effect on trabecular or cortical bone. Cortical bone loss and bone weakness were induced by glucocorticoids while they improved muscle grip strength in Mdx mice. 25-hydroxy D deficiency did not result in any significant effects on growing bone or muscle in the Mdx mice. In combination with glucocorticoid treatment, low 25-hydroxy D resulted in no change in cortical bone mineral density but bone ductility was significantly increased suggesting lower bone mineralization.

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp(-/-) mice given a single injection of 100 µg/kg dioxin did not survive beyond day 5; all Tiparp(+/+) mice survived the 30-day treatment. Dioxin-treated Tiparp(-/-) mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

Relative Contributions of CYP1A2 and CYP2E1 to the Bioactivation and Clearance of 4-Aminobiphenyl in Adult Mice.

4-Aminobiphenyl (ABP), a prototypical aromatic amine carcinogen in rodents and humans, requires bioactivation to manifest its toxic effects. A traditional model of ABP bioactivation, based on in vitro enzyme kinetic evidence, had postulated initial N-hydroxylation by the cytochrome P450 isoform CYP1A2. This is followed by phase 2 O-conjugation and hydrolysis to form a reactive nitrenium ion that covalently binds to DNA and produces tumor-initiating mutations. However, Cyp1a2(-/-) mice still possess significant liver ABP N-hydroxylation activity, DNA damage, and incidence of ABP-induced liver tumors, and in vivo induction of CYP1A2 paradoxically reduces levels of ABP-induced DNA damage. Competing ABP detoxification pathways can include N-acetylation by arylamine N-acetyltransferase 1 (NAT1) and/or NAT2; however, wild-type and Nat1/2(-/-) mice have similar in vivo ABP clearance rates. Together, these studies suggest the existence of novel ABP bioactivating and clearance/detoxification enzymes. In the present study, we detected similar reductions in Vmax for ABP N-hydroxylation by liver microsomes from Cyp1a2(-/-) and Cyp2e1(-/-) mice when compared with wild-type mice. In addition, recombinant mouse CYP1A2 and CYP2E1 were both able to N-hydroxylate ABP in mouse hepatoma cells. However, the in vivo clearance of ABP was significantly reduced in Cyp1a2(-/-) but not in Cyp2e1(-/-) mice. Our results support a significant role for CYP2E1 as a novel ABP N-oxidizing enzyme in adult mice, and suggest a more important contribution of CYP1A2 to the in vivo plasma clearance and thus detoxification of ABP.

Sex Differences in Metabolic and Behavioral Responses to Exercise but Not Exogenous Osteocalcin Treatment in Mice Fed a High Fat Diet.

Background: Exercise helps improve glucose handling in diabetes and has been shown to improve mood and cognition in other conditions. Osteocalcin, a protein produced by bone osteoblasts, was reported to have endocrine actions to improve both metabolism and also improve age-related cognitive deficits in mice. Methods: This study was designed to compare the effects of daily treadmill running exercise with injection of osteocalcin in high fat diet (HFD) induced diabetes in male and female C57BL/6J mice. Following established glucose intolerance and treatment for 8 weeks, mice were assessed for anxiety on an elevated plus maze, motivation by tail suspension test and cognition and memory in a puzzle box. Endogenous osteocalcin was measured by ELISA. Results: Mice on HFD had high weight gain, glucose intolerance and increased white fat. Exercise increased circulating osteocalcin levels in female mice but decreased them in male mice. Exercise also decreased weight gain and improved glucose tolerance in female but not male mice; however, treatment with osteocalcin made no metabolic improvements in either males or females. HFD induced anxiety only in female mice and this was not improved by osteocalcin. Exercise induced anxiety only in male mice. HFD also increased depressive-like behavior in both sexes, and this was improved by either exercise or osteocalcin treatment. Cognitive deficits were seen in both male and female mice on HFD. Exercise improved cognitive performance in female but not male mice, while osteocalcin treatment improved cognitive performance in both sexes. Conclusion: There were sex differences in the effects of exercise on endogenous osteocalcin regulation that correlated with improvements in cognitive but not metabolic outcomes. Exogenous osteocalcin did not improve metabolism but was effective in improving HFD-induced cognitive deficits. Sex is an important variable in hormonal and cognitive responses to exercise in diabetes.

Increased osteoblast GαS level determines bone response to hyperparathyroidism in female mice.

GS, the stimulatory heterotrimeric G protein, is an essential regulator of osteogenesis and bone turnover. To determine if increasing GαS in osteoblasts alters bone responses to hyperparathyroidism, we used a transgenic mouse line overexpressing GαS in osteoblasts (GS-Tg mice). Primary osteoblasts from GS-Tg mice showed increased basal and parathyroid hormone (PTH)-stimulated cAMP and greater responses to PTH than cells from WT mice. Skeletal responses to 2-week continuous PTH administration (cPTH) in female mice resulted in trabecular bone loss in WT mice but 74% and 34% increase in trabecular bone mass in long bones and vertebrae, respectively, in GS-Tg mice. Vertebral biomechanical strength was compromised by cPTH treatment in WT mice but not in GS-Tg. Increased peritrabecular fibrosis was greatly increased by cPTH in Gs-Tg compared to WT mice and corresponded with greater increases in Wnt pathway proteins in trabecular bone. Cortical bone responded negatively to cPTH in WT and Gs-Tg mice with large increases in porosity, decreased cortical thickness and compromised biomechanical properties. These results demonstrate that hyperparathyroidism can increase trabecular bone when GS expression and cAMP stimulation in osteoblasts are increased but this is not the case in cortical bone where increased GS expression exacerbates cortical bone loss.

ß2-adrenergic receptors inhibit the expression of collagen type II in growth plate chondrocytes by stimulating the AP-1 factor Jun-B.

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through ß(2)-adrenergic receptors (ß(2)-AR). We have shown previously that ß(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined ß(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of ß(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by ∼50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated ß(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through ß(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.

Liver-selective expression of human arylamine N-acetyltransferase NAT2 in transgenic mice.

Human arylamine N-acetyltransferase 2 (NAT2) mediates the biotransformation of arylamine drugs and procarcinogens into either innocuous or reactive DNA-damaging metabolites and is expressed predominantly in liver. Interspecies differences and incongruous results between in vitro, in vivo, and epidemiological studies make it difficult to extrapolate animal results to human risk. We have generated human NAT2 transgenic mice on both C57BL/6 (hNAT2(tg)) and Nat1/2 null backgrounds [hNAT2(tg)Nat1/2(-/-)], in which liver-selective expression of human NAT2 is driven by the mouse albumin promoter. We detected expression of the human NAT2 transcript and protein in mouse liver by real-time PCR and Western blot analysis. NAT2 enzyme activity, measured using the human NAT2-selective substrate sulfamethazine (SMZ), was 40- to 80-fold higher in liver cytosols from hNAT2(tg)Nat1/2(-/-) mice than in wild-type mice. An unexpected gender difference was observed, with males displaying 2-fold higher activity than females. Transgenic mice also had an increased in vivo plasma clearance of SMZ and higher levels of N-acetylated SMZ than wild-type mice. Liver expression of human NAT2 did not affect the disposition of the human NAT1-selective substrate p-aminosalicylic acid (PAS), because hNAT2(tg)Nat1/2(-/-) mice displayed in vivo PAS pharmacokinetic profiles similar to those of Nat1/2(-/-) mice. The metabolism of 4-aminobiphenyl was similar between hNAT2(tg)Nat1/2(-/-) and wild-type mice with the exception of a more liver-restricted pattern in hNAT2(tg)Nat1/2(-/-) mice and lower activity in females. Overall, the hNAT2(tg)Nat1/2(-/-) mouse mimics human expression of NAT2 and may thus be of value in clarifying the role of human NAT2 in arylamine clearance, detoxification, and bioactivation.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)-Inducible Poly-ADP-Ribose Polymerase (TIPARP/PARP7) Catalytic Mutant Mice (TiparpH532A) Exhibit Increased Sensitivity to TCDD-Induced Hepatotoxicity and Lethality.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.

A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models.

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, ß-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including ß-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

The impact of sex on hepatotoxic, inflammatory and proliferative responses in mouse models of liver carcinogenesis.

Liver cancer is the third most common cause of cancer-related death but is almost 4-fold more prevalent in men than in women. Increased risk in men may be due in part to elevated chronic inflammation, which is a crucial driving force for many cancers. Male mice also have a greater incidence of liver cancer than females after postnatal exposure to procarcinogens such as 4-aminobiphenyl (ABP) or diethylnitrosamine (DEN), or in mice that transgenically express hepatitis B virus (HBV) proteins. Liver damage, inflammation and proliferation are central to liver cancer development, and previous studies have shown that hepatocellular damage, inflammation and proliferation are acutely elevated to a greater extent in adult male mice than in females after high-dose exposure to DEN. In contrast, postnatal exposure of mice to tumor-inducing doses of either DEN or ABP produces no such acute responses. However, it is not known whether sex differences in responses to postnatal carcinogen exposure or to HBV protein expression may develop over time following sexual maturation. We conducted an extended time course study to compare markers of liver damage, inflammation and proliferation between male and female mice exposed postnatally to 600 nmol ABP or 10 mg/kg DEN, and also in HBV transgenic (HBVTg) mice, over the duration of time that mice are normally maintained for standard liver tumor development protocols. Postnatal exposure to either ABP or DEN produced no evidence of either acute or chronic hepatocyte damage, liver inflammation or proliferation in either male or female mice. In contrast, HBVTg mice showed increased liver damage, inflammation and proliferation with age, but with no observed sex difference. These findings suggest that although chronic liver damage, inflammation and proliferation may be drivers for liver cancer development, they are unlikely to contribute directly to observed sex differences in liver tumor risk.

Endostatin as a Mediator Between Endothelial Function and Cognitive Performance in Those at Risk for Vascular Cognitive Impairment.

BACKGROUND: Patients with coronary artery disease have an increased risk for developing vascular cognitive impairment. Endothelial function is often diminished and has been associated with lower cognitive performance in these patients. The link between endothelial function and cognition in coronary artery disease is not fully understood. Angiogenesis may play a role in mediating the association between endothelial function and cognition since angiogenic processes rely heavily on the endothelium. OBJECTIVE: The aim of this study was to determine if markers of angiogenesis mediate the relationship between endothelial function and cognition in coronary artery disease patients. METHODS: In 50 participants with coronary artery disease, endothelial function was assessed using peripheral arterial tonometry. Vascular endothelial growth factor (pro-angiogenic) and endostatin (anti-angiogenic) were measured in peripheral serum samples. Cognition was assessed using the Montreal Cognitive Assessment. A mediation analysis, using a bias corrected inferential bootstrapping method with 10,000 permutations, was used to determine if vascular endothelial growth factor or endostatin mediated an association between peripheral arterial tonometry measures and cognitive performance on the Montreal Cognitive Assessment. RESULTS: Endostatin, but not vascular endothelial growth factor, mediated a relationship between endothelial function and cognitive performance when controlling for total years of education, body mass index, coronary artery bypass graft, stent, diabetes, and diuretic use. This analysis was also significant when delayed recall was substituted for the overall score on the Montreal Cognitive Assessment. CONCLUSION: These results suggest that endostatin mediates an association between endothelial function and cognitive performance in coronary artery disease.

Serum osteocalcin is associated with subjective stress in people with depression and type 2 diabetes.

BACKGROUND: Low serum osteocalcin is a risk factor for type 2 diabetes mellitus (T2DM), and osteocalcin release from bone is associated with an acute stress response in mice. Both diabetes and stress are associated with depression. Here, we assess relationships between serum osteocalcin, depression and subjective stress in people with T2DM. METHODS: Participants with T2DM (HbA1c above 6.4 %, impaired fasting glucose or impaired glucose tolerance) were assessed for a major depressive episode using the research version of the Structured Clinical Interview for DSM-5 depression criteria (SCID-5RV). Subjective stress over the past month was assessed using the Perceived Stress Scale (PSS). Serum carboxylated (cOCN) and fully decarboxylated (dcOCN) osteocalcin were assayed from fasting morning blood by commercial enzyme-linked immunosorbent assay. RESULTS: Among 95 participants (mean age 62.4 ± 9.9, 51 % women), 22 % were experiencing a depressive episode (9 men, 12 women). The presence of a depressive episode was not associated with dcOCN or cOCN concentrations; however, higher concentrations of cOCN were associated with higher PSS scores in participants with depression (r = 0.585, p = 0.005). In an analysis of covariance model controlling for age, sex, body mass index, glycemic control (glycosylated hemoglobin), insulin resistance (homeostatic model), depression, and antidepressant use, cOCN was associated with PSS scores (F=10.302, p = 0.002), and this relationship was stronger in those with depression (depression × cOCN interaction F=4.978, p = 0.028). Although associations between dcOCN concentrations and PSS scores did not reach significance, the same trend seen with cOCN concentrations was observed in participants with depression for dcOCN (r=0.365, p=0.10), and for a depression × dcOCN interaction associated with PSS scores in the whole group (F=2.165, p = 0.15). CONCLUSIONS: Osteocalcin is a neuroendocrine marker associated with perceived chronic stress among people with T2DM experiencing a depressive episode.

Primary aromatic amines and cancer: Novel mechanistic insights using 4-aminobiphenyl as a model carcinogen.

Aromatic amines are an important class of human carcinogens found ubiquitously in our environment. It is estimated that 1 in 8 of all known or suspected human carcinogens is or can be converted into an aromatic amine, making the elucidation of their mechanisms of toxicity a top public health priority. Decades of research into aromatic amine carcinogenesis revealed a complex bioactivation process where Phase I and Phase II drug metabolizing enzymes catalyze N-oxidation and subsequent conjugation reactions generating the highly electrophilic nitrenium intermediate that reacts with and forms adducts on cellular macromolecules. Although aromatic amine-DNA adducts were believed to be the main driver of cancer formation, several studies have reported a lack of correlation between levels of DNA adducts and tumors. Using genetically modified mouse models, our laboratory and others observed several instances where levels of conventionally measured DNA adducts failed to correlate with liver tumor incidence following exposure to the model aromatic amine procarcinogen 4-aminobiphenyl. In this review we first provide a historical overview of the studies that led to a proposed mechanism of carcinogenesis caused by aromatic amines, where their bioactivation to form DNA adducts represents the central driver of this process. We then highlight recent mechanistic studies using 4-aminobiphenyl that are inconsistent with this mechanism which suggest novel drivers of aromatic amine carcinogenesis.

A readout of metabolic efficiency in arylamine N-acetyltransferase-deficient mice reveals minor energy metabolism changes.

Recent studies have revealed a possible link between the activities of polymorphic arylamine N-acetyltransferases (NATs) and energy metabolism. We used a Nat1/Nat2 double knockout (KO) mouse model to demonstrate that ablation of the two Nat genes is associated with modest, intermittent alterations in respiratory exchange rate. Pyruvate tolerance tests show that double KO mice have attenuated hepatic gluconeogenesis when maintained on a high-fat/high-sucrose diet. Absence of the two Nat genes also leads to an increase in the hepatic concentration of coenzyme A in mice fed a high-fat/high-sucrose diet. Our results suggest a modest involvement of NAT in energy metabolism in mice, which is consistent with the absence of major phenotypic deregulation of energy metabolism in slow human acetylators.

Overexpression of GαS in Murine Osteoblasts In Vivo Leads to Increased Bone Mass and Decreased Bone Quality.

GαS is a heterotrimeric G protein that transduces signals from activated G protein-coupled receptors on the cell surface to stimulate adenylyl cyclase/cyclic adenosine monophosphate (AMP) signaling. GαS plays a central role in mediating numerous growth and maintenance processes including osteogenesis and bone turnover. Decreased GαS expression or activating mutations in GαS both affect bone, suggesting that modulating GαS protein levels may be important for bone health and development. To examine the effects of increased osteoblastic GαS expression on bone development in vivo, we generated transgenic mice with GαS overexpression in osteoblasts (HOM-Gs mice) driven by the 3.6-kilobase (kb) Col1A1 promoter. Both male and female HOM-Gs mice exhibit increased bone turnover with overactive osteoblasts and osteoclasts, resulting in a high bone mass phenotype with significantly reduced bone quality. At 9 weeks of age, HOM-Gs mice have increased trabecular number, volumetric BMD (vBMD), and bone volume; however, the bone was woven and disorganized. There was also increased cortical bone volume despite an overall reduction in size in HOM-Gs mice along with increased cortical porosity and brittleness. The skeletal phenotype of HOM-Gs mice progressed into maturity at 26 weeks of age with further accrual of trabecular bone, whereas WT mice lost trabecular bone at this age. Although cortical bone volume and geometry were similar between mature HOM-Gs and WT mice, increased porosity persisted and the bone was weaker. At the cellular level, these alterations were mediated by an increase in bone resorption by osteoclasts and an overwhelmingly higher increase in bone formation by osteoblasts. In summary, our findings demonstrate that high osteoblastic GαS expression results in aberrant skeletal development in which bone production is favored at the cost of bone quality. © 2017 American Society for Bone and Mineral Research.

Deficiency of N-acetyltransferase increases the interactions of isoniazid with endobiotics in mouse liver.

Acetylation is the major metabolic pathway of isoniazid (INH) mediated by N-acetyltransferases (NATs). Previous reports suggest that slow acetylators have higher risks of INH hepatotoxicity than rapid acetylators, but the detailed mechanisms remain elusive. The current study used Nat1/2(-/-) mice to mimic NAT slow metabolizers and to investigate INH metabolism in the liver. We found that INH acetylation is abolished in the liver of Nat1/2(-/-) mice, suggesting that INH acetylation is fully dependent on NAT1/2. In addition to the acetylation pathway, INH can be hydrolyzed to form hydrazine (Hz) and isonicotinic acid (INA). We found that INA level was not altered in the liver of Nat1/2(-/-) mice, indicating that deficiency of NAT1/2 has no effect on INH hydrolysis. Because INH acetylation was abolished and INH hydrolysis was not altered in Nat1/2(-/-) mice, we expected an extremely high level of INH in the liver. However, we only observed a modest accumulation of INH in the liver of Nat1/2(-/-) mice, suggesting that there are alternative pathways in INH metabolism in NAT1/2 deficient condition. Our further studies revealed that the conjugated metabolites of INH with endobiotics, including fatty acids and vitamin B6, were significantly increased in the liver of Nat1/2(-/-) mice. In summary, this study illustrated that deficiency of NAT1/2 decreases INH acetylation, but increases the interactions of INH with endobiotics in the liver. These findings can be used to guide future studies on the mechanisms of INH hepatotoxicity in NAT slow metabolizers.

Positive effects of bisphosphonates on bone and muscle in a mouse model of Duchenne muscular dystrophy.

Patients with Duchenne muscular dystrophy are at increased risk of decreased bone mineral density and bone fracture as a result of inactivity. To determine if antiresorptive bisphosphonates could improve bone quality and their effects on muscle we studied the Mdx mouse, treated with pamidronate during peak bone growth at 5 and 6 weeks of age, and examined the outcome at 13 weeks of age. Pamidronate increased cortical bone architecture and strength in femurs with increased resistance to fracture. While overall long bone growth was not affected by pamidronate, there was significant inhibition of remodeling in metaphyseal trabecular bone with evidence of residual calcified cartilage. Pamidronate treatment had positive effects on skeletal muscle in the Mdx mice with decreased serum and muscle creatine kinase and evidence of improved muscle histology and grip strength.