Smad-3 as a mediator of the fibrotic response

Introduction Smad‐3, a key cytoplasmic mediator of transforming growth factor‐β (TGF‐β) signalling, mediates many of its inflammatory and fibrotic effects in vivo ( Roberts et al. 2001 ). Smad‐3 null mice are protected against cutaneous injury induced by ionizing irradiation ( Flanders et al. 2002 ). Here, we report on our continuing studies on radioprotection as well as protection against tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in Smad‐3 null mice. Methods For radioprotection studies, the flank skin of Smad‐3^+/+ wild‐type (WT) and Smad‐3^–/– knockout (KO) mice was exposed to 30 Gy of localized γ‐irradiation and analysed for histology and gene expression at various times post irradiation. In the UUO model, the right proximal ureter of WT and KO mice was ligated, and 1–2 weeks later kidneys were analysed for inflammation, fibrosis and gene expression. Results Six weeks after exposure to irradiation, skin from KO mice shows less epidermal acanthosis and influx of mast cells, macrophages and neutrophils than skin of WT mice. Paradoxically, at 6–8 h post irradiation, KO skin shows a significantly greater number of neutrophils. Irradiated KO skin also exhibits less immunoreactive TGF‐β, fewer myofibroblasts and less scarring than does WT. Smad‐3 null dermal fibroblasts do not respond to the chemotactic effects of TGF‐β and show less induction of fibrogenic cytokines when treated with irradiation plus TGF‐β compared to WT cells. Following UUO, normal kidney architecture is preserved in KO mice, while kidneys from WT mice are enlarged with an influx of mononuclear cells and increased expression of collagen and TGF‐β1. Additionally, renal tubules in obstructed kidneys of KO mice remain positive for E‐cadherin without expression of α‐smooth muscle actin, while the opposite expression pattern is seen in obstructed kidneys of WT mice. TGF‐β treatment of primary cultures of WT renal tubular epithelial cells results in a phenotypic change from a cobblestone pattern to a spindle‐shaped fibroblastic appearance, while KO cells treated with TGF‐β maintain their original appearance. Conclusion Smad‐3 plays an important role in mediating pathogenic inflammation and fibrosis in several model systems and is also essential for TGF‐β1‐induced epithelial–mesenchymal transition in renal tubular epithelial cells. Inhibitors of the Smad‐3 pathway may have clinical applications in the treatment of a number of fibrotic conditions.     

A model of sensitivity: 1,3-butadiene increases mutant frequencies and genomic damage in mice lacking a functional microsomal epoxide hydrolase gene

The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO(2)]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO(2) (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P < 0.05) 12.4-fold increase in MF over controls and a significant 5.4-fold increase in MF over exposed WT mice. Additionally, KO mice exposed to BDO(2) exhibited a significant 4.5-fold increase in MF over controls and a significant 1.7-fold increase in MF over exposed WT mice. We also compared genomic damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO(2). KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5-12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO(2) exhibited significantly more DNA damage than all other groups (2.3-27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r(2) = 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO(2). This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD.     

IL-6 deficiency exacerbates skin inflammation in a murine model of irritant dermatitis

Contact dermatitis is the second most reported occupational injury associated with workers compensation. Inflammatory cytokines are closely involved with the development of dermatitis, and their modulation could exacerbate skin damage, thus contributing to increased irritancy. IL-6 is a pro-inflammatory cytokine paradoxically associated with both skin healing and inflammation. To determine what role this pleiotropic cytokine plays in chemically-induced irritant dermatitis, IL-6 deficient (KO), IL-6 over-expressing transgenic (TgIL6), and corresponding wild-type (WT) mice were exposed to acetone or the irritants JP-8 jet fuel or benzalkonium chloride (BKC) daily for 7 days. Histological analysis of exposed skin was performed, as was tissue mRNA and protein expression patterns of inflammatory cytokines via QPCR and multiplex ELISA. The results indicated that, following JP-8 exposure, IL-6KO mice had greatly increased skin IL-1β, TNFα, CCL2, CCL3, and CXCL1 mRNA and corresponding product protein expression when compared to that of samples from WT counterparts and acetone-exposed control mice. BKC treatment induced the expression of all cytokines examined as compared to acetone, with CCL2 significantly higher in skin from IL-6KO mice. Histological analysis showed that IL-6KO mice displayed significantly more inflammatory cell infiltration as compared to WT and TgIL6 mice in response to jet fuel. Analysis of mRNA for the M2 macrophage marker CD206 indicated a 4-fold decrease in skin of IL-6KO mice treated with either irritant as compared to WT. Taken together, these observations suggest that IL-6 acts in an anti-inflammatory manner during irritant dermatitis, and these effects are dependent on the chemical nature of the irritant.     

Interleukin-6 modifies mRNA expression in mouse skeletal muscle

Aim: The aim of this study was to test the hypothesis that interleukin (IL)‐6 plays a role in exercise‐induced peroxisome proliferator‐activated receptor γ co‐activator (PGC)‐1α and tumor necrosis factor (TNF)‐α mRNA responses in skeletal muscle and to examine the potential IL‐6‐mediated AMP‐activated protein kinase (AMPK) regulation in these responses. Methods: Whole body IL‐6 knockout (KO) and wildtype (WT) male mice (4 months of age) performed 1 h treadmill exercise. White gastrocnemius (WG) and quadriceps (Quad) muscles were removed immediately (0′) or 4 h after exercise and from mice not run acutely. Results: Acute exercise reduced only in WT muscle glycogen concentration to 55 and 35% (P < 0.05) of resting level in Quad and WG respectively. While AMPK and Acetyl CoA carboxylase (ACC) phosphorylation increased 1.3‐fold (P < 0.05) in WG and twofold in Quad immediately after exercise in WT mice, no change was detected in WG in IL‐6 KO mice. The PGC‐1α mRNA content was in resting WG 1.8‐fold higher (P < 0.05) in WT mice than in IL‐6 KO mice. Exercise induced a delayed PGC‐1α mRNA increase in Quad in IL‐6 KO mice (12‐fold at 4 h) relative to WT mice (fivefold at 0′). The TNF‐α mRNA content was in resting Quad twofold higher (P < 0.05) in IL‐6 KO than in WT, and WG TNF‐α mRNA increased twofold (P < 0.05) immediately after exercise only in IL‐6 KO. Conclusion: In conclusion, IL‐6 affects exercise‐induced glycogen use, AMPK signalling and TNF‐α mRNA responses in mouse skeletal muscle.     

Ovarian morphometrics in TP53‐deficient mice

The objective of these investigations was to characterize ovarian responses to hormonal stimulation in TP53‐deficient mice. TP53‐deficient (KO) and wild‐type (WT) mice were induced to ovulate with pregnant mare serum gonadotropin followed by human chorionic gonadotropin. Effect of estradiol on ovarian morphology was determined in induced and control mice implanted with estradiol‐containing or placebo pellets. Blood was collected and mice were killed 7 days following implantation. Preserved ovaries were serially sectioned and stained. Numbers of follicles (all classifications) decreased with ovulation induction, but did not differ between WT and KO mice. Numbers of corpora lutea (CL) were less in ovulation‐induced KO mice treated with estradiol compared to WT mice. Area of individual CL and serum concentrations of progesterone were greater in ovulation‐induced KO mice given estradiol compared to WT mice. Ovulation‐induced KO mice had more, larger hemorrhagic follicles than similarly treated WT mice, but hemorrhagic follicles were not influenced by estradiol. Proliferation of ovarian surface epithelial cells did not differ between KO and WT mice induced to ovulate and given estradiol. Ovaries from TP53 gene knockout mice (n = 4) induced to ovulate and given a 21‐day estradiol implant three times over 58 days were observed for precursor lesions. There was no indication of precursor lesions in any TP53 KO or WT mouse. TP53 status did not influence recruitment of follicles, but TP53 deficiency hindered the ability of human chorionic gonadotropin to cause ovulation. Anat Rec, 290:59–64, 2007. © 2006 Wiley‐Liss, Inc.     

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle‐Specific VEGF Expression

Vascular endothelial growth factor‐A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle‐specific VEGF knockout (mVEGF‐KO, n = 7) mice and their wild‐type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: ?63.5%; soleus: ?54.8%; P ≤ 0.05) in mVEGF‐KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv‐values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF‐KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse‐strains: Vv (–17.4%), absolute area size (–19.1%) and thickness (–19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF‐KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF‐KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239–2249, 2017. © 2017 Wiley Periodicals, Inc.     

Methyl isocyanate: an evaluation of in vivo cytogenetic activity

The ability of inhaled methyl isocyanate (MIC) to induce genotoxic and cytotoxic damage in vivo was evaluated by assessing the induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in bone marrow metaphase cells, the induction of micronuclei in polychromatic erythrocytes (MN-PCEs), and the inhibition of bone marrow cellular proliferation and erythropoiesis. B6C3F1 mice were exposed to MIC by two exposure regiments: in two experiments, male mice only were exposed to 3, 10, and 30 ppm for 2 hr; in four experiments, male and female mice were exposed to 1 and 3 ppm (in one experiment, to 6 ppm, also), 6 hr per day for 4 consecutive days. The various cytogenetic endpoints were analyzed in bone marrow and peripheral blood (4-day exposure regimen only) samples taken from bromodeoxyuridine tablet-implanted animals killed 11 to 22 hr after cessation of the exposure to MIC. Exposure to MIC for 2 hr induced a significant delay in cellular proliferation but did not induce a significant increase in CAs, SCEs (evaluated at 3 and 10 ppm, only) or in bone marrow MN-PCEs. Also, this exposure regimen did not inhibit the rate of erythropoiesis. Following exposure to MIC for 4 days, a weak but significant increase in CAs and SCEs was observed in male (in one experiment) and in female (in two experiments) mice. The induction was especially apparent in the single experiment in which mice were exposed to 6 ppm MIC. At this concentration, a significant increase in MN-PCEs in peripheral blood was observed in male but not female mice. Delay in bone marrow cell proliferation was observed in male mice beginning at 3 ppm and in female mice at 6 ppm. The 4-day exposure regimen resulted also in a depressed rate of erythropoiesis, with male mice appearing to exhibit greater depression than female mice. The results demonstrate that exposure to MIC by inhalation results in bone marrow damage, indicating the systemic genotoxic/cytotoxic activity of MIC and/or reactive metabolites.     

The effect of interleukin-4 on the induction of intestinal mast cells and chronological cytokine profiles during intestinal nematode Strongyloides ratti infection

The effect of interleukin-4 (IL-4) on the induction of intestinal mast cells and cytokine profiles during Strongyloides ratti infection was studied using IL-4 knockout (IL-4 KO) mice. The antigen-specific proliferative response of mesenteric lymph node cells was not impaired in IL-4 KO mice. The number of intestinal mast cells induced in IL-4 KO mice during S. ratti infection was 2- to 3-fold lower than that observed in WT mice. Intestinal mastocytosis had disappeared in IL-4 KO mice by day 21 postinfection, when significant mastocytosis continued to be observed in WT mice. In mesenteric lymphnode of IL-4 KO, IL-3 production decreased and mice IFN-γ production significantly increased as compared with those of WT mice. The numbers of eggs excreted per gram of feces (EPG) by IL-4 KO mice were greater than those excreted by WT mice on day 6 postinfection, but no difference was observed in the subsequent period. In conclusion, intestinal mast cells are induced during S. ratti infection in the absence of IL-4, and IL-4 is not essential for protection against intestinal adult worms of S. ratti. Received: 27 June 2000 / Accepted: 6 July 2000     

Suppression of antigen-specific antibody responses in mice exposed to perfluorooctanoic acid: Role of PPARα and T- and B-cell targeting

T-cell-dependent antibody responses (TDAR) are suppressed in female C57BL/6N mice exposed to ≥3.75?mg/kg of perfluorooctanoic acid (PFOA) for 15 days. To determine if suppression of humoral immunity by PFOA is peroxisome proliferator activated receptor alpha (PPARα)-dependent and if suppression is associated with specific targeting of T- or B-cells, three separate experiments were conducted: (1) female PPARα constitutive knockout (PPARα KO; B6.129S4-Ppar^tm1GonzN12) and wild-type controls (WT; C57BL/6-Tac) exposed to 0, 7.5, or 30?mg PFOA/kg for 15 days were immunized on Day 11 with a T-cell-dependent antigen and sera then collected for measures of antigen-specific IgM titers (TDAR) 5 days later; (2) female C57BL/6N WT mice exposed to 0, 0.94, 1.88, 3.75, or 7.5?mg PFOA/kg for 15 days were immunized with a T-cell-independent antigen on Day 11 and sera were then collected for analyses of antigen-specific IgM titers (TIAR) 7 days later; and (3) splenic lymphocyte phenotypes were assessed in unimmunized female C57BL/6N WT mice exposed to 0, 3.75, or 7.5?mg PFOA/kg for 10 days to investigate effects of PFOA in the absence of specific immunization. Separate groups of mice were immunized with a T-cell-dependent antigen after 11 days of exposure and splenic lymphocyte sub-populations were assessed after 13 or 15 days of exposure to assess numbers of stimulated cells. The results indicated that exposure to ≥1.88?mg PFOA/kg suppressed the TIAR; exposure to 30?mg PFOA/kg suppressed the TDAR in both PPARα KO and WT mice. The percentage of splenic B-cells was unchanged. Results obtained in the PPARα KO mice indicated that PPARα suppression of TDAR was independent of PPARα involvement. Suppression of the TIAR and the TDAR with minimal lymphocyte sub-population effects suggested that effects on humoral immunity are likely mediated by disruption of B-cell/plasma cell function.     

Catalytic and non‐catalytic roles of DNA polymerase κ in the protection of human cells against genotoxic stresses

DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm‐6‐MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild‐type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non‐catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non‐catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed. Environ. Mol. Mutagen. 56:650–662, 2015. © 2015 Wiley Periodicals, Inc.     

Assessment of carcinogenicity of di(2-ethylhexyl)phthalate in a short-term assay using Xpa-/- and Xpa-/-/p53+/- mice

The potential of Xpa-/- and Xpa-/-/p53+/- mice for short-term carcinogenicity assays was evaluated with di(2-ethylhexyl)phthalate (DEHP). Groups of 15 male and female Xpa-/- mice, received diets containing 0, 1, 500, 3,000, or 6,000 ppm DEHP, and wild-type (WT) and Xpa-/-p53+/-mice 0 or 6,000 ppm DEHP for 39 weeks. Xpa-/-, Xpa-/-/p53+/-, and WT males, fed 2,500 ppm p-cresidine, served as a positive control. In all models, the survival was not altered by DEHP. Increased incidences of nonneoplastic lesions were recorded in testes and kidneys with no apparent difference between the models. The only liver tumors in all models were adenomas in males with no statistically significantly increased incidence. For p-cresidine. the survival was decreased (p < 0.05) only in transgenic models. Statistically significantly increased incidences of nonneoplastic lesions were recorded in the liver, urinary bladder, and nasal cavity in all models, and in kidneys in transgenic models. The only tumors with statistically significantly increased incidence were liver adenomas in transgenic models (XPA: I vs 7: 'XPA/p53': 0 vs 12; WT: 0 vs 5, p = 0.053) and urinary bladder carcinomas in XPA/p53 model (0 vs 7). The negative carcinogenic response to DEHP and the positive response to p-cresidine support the expected sensitivity to genotoxic carcinogens in these transgenic models.     

Outcome of acetylcholinesterase deficiency for neuromuscular functioning

Acetylcholinesterase (AChE) plays an essential role in neuromuscular transmission, therefore it is surprising that AChE knockout (KO) mice could live to the adulthood. Neuromuscular functioning in KO and normal (wild type, WT) mice were studied, at different age (1.5-, 4- and 9-month-old). Hindlimb muscle force productions in response to nerve or muscle electric stimulation were recorded in situ and in vitro. Our results show that contrary to WT mice, 1.5-, 4- and 9-month-old KO mice exhibited a decreased in tetanic force during short periods (500 ms) of repetitive nerve stimulations (tetanic fade). Nevertheless submaximal muscle forces in response to single or repetitive nerve stimulation were increased (potentiation) in 1.5-, 4- and 9-month-old KO mice as compared to WT mice (p<0.05). Tetanic fade and potentiation were absent when muscles were directly stimulated, indicating neuromuscular transmission alterations in KO mice. Contrary to younger mice, muscle weight and maximal tetanic force in response to repetitive nerve stimulation were not reduced in 4- and 9-month-old KO mice as compared to WT mice (p>0.05). In conclusion AChE deficit leads to marked neuromuscular alterations in hind limb muscle functioning and a prominent symptom is the lack of resistance to fatigue.     

cytogenetic and germ cell effects of phosphine inhalation by rodents: II. subacute exposures to rats and mice

Phosphine (PH₃) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH₃, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH₃ for 6 hr/day for 9 days over an 11 -day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH₃ up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11 -day period as measured by several cytogenetic assays. To evaluate the effects of PH₃ on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH₃ for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH₃ by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm. © 1994 Wiley-Liss, Inc.     

The oestrogen receptor β contributes to sex related differences in endothelial function of murine small arteries via EDHF

Sex related differences in cardiovascular function have been reported in oestrogen receptor β knockout (ERβKO) mice. In this study we examined the role of endothelium-derived hyperpolarizing factor (EDHF) in differences in small artery endothelial function between ERβKO and wild-type (WT) mice. Small femoral arteries were isolated from ERβKO and WT mice and mounted on a wire myograph. Concentration–response curves to ACh were compared before and after incubation with inhibitors of nitric oxide (NO) and prostacyclin (PGI2) synthesis. Comparison of the expression of the principal vascular connexins (Cx37, 40 and 43), implicated in EDHF-mediated dilatation were undertaken by immunohistochemistry. Vascular ultrastructure was studied by transmission and scanning electron microscopy. ACh-induced relaxation of arteries (< 200 μm internal diameter) was greater in WT females versus males and was attributable to a greater EDHF component of relaxation. This sex difference was absent in ERβKO mice. Arteries from ERβKO males (but not females) were more sensitive to ACh compared to WT. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF-mediated responses was confirmed in male arteries. The absence of ERβ had no influence on expression of main Cx subtypes within vascular wall or on ultrastructure and morphology of the endothelium. The data suggest that in WT male mice, ERβ reduces EDHF-mediated relaxation through gap junction communication.     

Hepcidin knockout mice spontaneously develop chronic pancreatitis owing to cytoplasmic iron overload in acinar cells

Iron is both an essential and a potentially toxic element, and its systemic homeostasis is controlled by the iron hormone hepcidin. Hepcidin binds to the cellular iron exporter ferroportin, causes its degradation, and thereby diminishes iron uptake from the intestine and the release of iron from macrophages. Given that hepcidin‐resistant ferroportin mutant mice show exocrine pancreas dysfunction, we analysed pancreata of aging hepcidin knockout (KO) mice. Hepcidin and Hfe KO mice were compared with wild‐type (WT) mice kept on standard or iron‐rich diets. Twelve‐month‐old hepcidin KO mice were subjected to daily minihepcidin PR73 treatment for 1 week. Six‐month‐old hepcidin KO mice showed cytoplasmic acinar iron overload and mild pancreatitis, together with elevated expression of the iron uptake mediators DMT1 and Zip14. Acinar atrophy, massive macrophage infiltration, fatty changes and pancreas fibrosis were noted in 1‐year‐old hepcidin KO mice. As an underlying mechanism, 6‐month‐old hepcidin KO mice showed increased pancreatic oxidative stress, with elevated DNA damage, apoptosis and activated nuclear factor‐κB (NF‐κB) signalling. Neither iron overload nor pancreatic damage was observed in WT mice fed iron‐rich diet or in Hfe KO mice. Minihepcidin application to hepcidin KO mice led to an improvement in general health status and to iron redistribution from acinar cells to macrophages. It also resulted in decreased NF‐κB activation and reduced DNA damage. In conclusion, loss of hepcidin signalling in mice leads to iron overload‐induced chronic pancreatitis that is not seen in situations with less severe iron accumulation. The observed tissue injury can be reversed by hepcidin supplementation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Lack of UDS activity in the livers of mice and rats exposed to dichloromethane

Dichloromethane (DCM) has been evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the livers of male mice and rats in vivo. Two types of experiment were conducted. In the first, Alpk:AP rats were exposed by oral gavage to 100, 500, or 1,000 mg/kg DCM and hepatocytes assessed for UDS via autoradiography 4 and 12 hours later. In the second, Fischer F344 rats or B6C3F1 mice were exposed by inhalation to either 2,000 or 4,000 ppm of DCM for either 2 or 6 hours, and hepatocytes assessed for UDS immediately after exposure. The dose levels and strains of rodent employed in the latter protocol correspond to those employed in a recent cancer bioassay of DCM conducted by the U.S. National Toxicology Program. DCM failed to induce UDS in any of the experiments. These data are discussed within the context of other evidence indicating DCM to be nongenotoxic in vivo, despite its reported carcinogenicity in the mouse.     

Ethylene oxide dose and dose-rate effects in the mouse dominant-lethal test

In the dose-response study, male mice were exposed by inhalation to ethylene oxide (EtO) for 4 consecutive days. Mice were exposed for 6 hr per day to 300 ppm, 400 ppm, or 500 ppm EtO for a daily total of 1,800, 2,400, or 3,000 ppm X hr (total exposures of 7,200, 9,600 and 12,000 ppm X hr), respectively. In the dose-rate study, mice were given a total exposure of 1,800 ppm X hr per day, also for 4 consecutive days, delivered either at 300 ppm in 6 hr, 600 ppm in 3 hr, or 1,200 ppm in 1.5 hr. Quantitation of dominant-lethal responses was made on matings involving sperm exposed as late spermatids and early spermatozoa, the most sensitive stages to EtO. In the dose-response study, a dose-related increase in dominant-lethal mutations was observed, the dose-response curve proved to be nonlinear. In the dose-rate study, increasing the exposure concentrations resulted in increased dominant-lethal responses.     

Integrated approach to the in vivo genotoxic effects of a titanium dioxide nanomaterial using LacZ plasmid‐based transgenic mice

Titanium dioxide (TiO₂) nanomaterials (NMs) are widely used in a diversity of products including cosmetics, pharmaceuticals, food, and inks, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. Previous studies on the genotoxicity of TiO₂ have reported discrepant or inconclusive findings in both in vitro and in vivo systems. This study explores the in vivo genotoxic potential of a well‐characterized uncoated TiO₂ NM with an average diameter of 22 nm (NM‐102, from JRC repository) using several genotoxicity endpoints in the LacZ plasmid‐based transgenic mouse model. Mice were exposed by intravenous injection to two daily doses of NM‐102: 10 and 15 mg/kg of body weight/day. Micronuclei were analyzed in peripheral blood reticulocytes 42 hr after the last treatment. DNA strand breaks (comet assay) and gene mutations were determined in the spleens and livers of the same animals 28 days after the last treatment. Histopathological and cytological analyses were also performed in liver samples. Genotoxic effects were not detected in mice exposed to the nanosized TiO₂ under the experimental conditions used, despite a moderate inflammatory response that was observed in the liver. Considering the biopersistence of TiO₂ in mouse liver and the moderate inflammatory response, the possibility of a secondary genotoxic effect at higher doses and in conditions that result in a stronger inflammatory response, for example, within a longer time window, should be investigated further. Environ. Mol. Mutagen. 55:500–509, 2014. © 2014 Wiley Periodicals, Inc.     

Colonization‐induced protection against invasive pneumococcal disease in mice is independent of CD103 driven adaptive immune responses

Nasopharyngeal colonization with Streptococcus pneumoniae (the pneumococcus) is known to mount protective adaptive immune responses in rodents and humans. However, the cellular response of the nasopharyngeal compartment to pneumococcal colonization and its importance for the ensuing adaptive immune response is only partially defined. Here we show that nasopharyngeal colonization with S. pneumoniae triggered substantial expansion of both integrin αE (CD103) positive dendritic cells (DC) and T lymphocytes in nasopharynx, nasal‐associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) of WT mice. However, nasopharyngeal de‐colonization and pneumococcus‐specific antibody responses were similar between WT and CD103 KO mice or Batf3 KO mice. Also, naïve WT mice passively immunized with antiserum from previously colonized WT and CD103 KO mice were similarly protected against invasive pneumococcal disease (IPD). In summary, the data show that CD103 is dispensable for pneumococcal colonization‐induced adaptive immune responses in mice.     

微管相关蛋白1B在FMR1基因敲除小鼠睾丸间质细胞中的表达

目的对不同周龄的Fmr1基因敲除和野生型雄性小鼠睾丸组织微管相关蛋白1B的表达进行分析比较,探讨Fmr1基因敲除小鼠的睾丸间质细胞微管相关蛋白1B表达的差异。方法采用不同周龄(4、6、8、10周)的FMR1基因敲除型(KO)和野生型(WT)各6只,先采用聚合酶链式反应(PCR)技术对KO小鼠和WT小鼠进行基因型鉴定,之后所有小鼠麻醉取睾丸组织、石蜡包埋切片进行HE染色对比观察Fmr1小鼠睾丸组织的形态,最后用免疫组织化学染色技术对KO小鼠和WT小鼠睾丸微管相关蛋白1B的表达进行检测并作对比分析。结果微管相关蛋白1B在4～10周小鼠睾丸间质细胞阳性表达,8、10周为强阳性表达,且KO小鼠在睾丸的阳性表达均高于WT小鼠。结论微管相关蛋白1B在同周龄FMR1基因敲除小鼠睾丸间质细胞的表达均显著高于WT小鼠,提示微管相关蛋白1B可能参与脆性X综合征巨睾症的发病过程。