Eumelanin is a major determinant for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) incorporation into hair of mice

Mice with different hair pigmentation were studied to evaluate the role of melanin in the incorporation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair. Mice C57BL/6J-c2j/+ (white), C57BL/6J-Ay (yellow), C57L/J (grey), C57BR/cdJ (brown) and C57BL/6J (black) were dosed with PhIP: 7-9 days old (total amount: 0.006 or 0.58 mg/kg b.wt., for 4 days) and adults (total amount 50 mg/kg b.wt. during 8 weeks). Hair was collected either 30 days after the last PhIP administration (new-born mice) or 8 weeks after the first administration (adult mice). PhIP was incorporated into black hair to a greater extent than into brown, grey, yellow and non-pigmented hair. The concentration of PhIP in the hair of new-born mice exposed to 0.58 mg PhIP/kg b.wt. were (mean+/-S.D.): 328+/-135 (black), 134+/-41 (brown), 9.1+/-1.2 (yellow) and 5.2+/-1.4 (white) ng/g hair. The PhIP concentrations in the hair of adult mice exposed to 50 mg/kg b.wt. were: 4750+/-1449 (black), 810+/-235 (brown), 541+/-119 (grey), 35.5+/-4.6 (yellow) and 21.6+/-8.8 (white) ng/g, and the eumelanin hair concentration in the same animals decreased in a similar pattern. A linear relationship (r2= 1.00, P<0.0001) between the relative PhIP incorporation and the eumelanin concentration in hair was found.     

Incorporation of the Food Mutagen 2‐Amino‐1‐Methyl‐6‐Phenylimidazo[4,5‐b]Pyridine (PhIP) into Fur and Correlation with Intestinal Tumourigenesis in Min/+ Mice

Abstract: The purpose of this work was to correlate the amount of the food mutagen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) determined in mouse fur with the number of intestinal tumours induced by PhIP, to further evaluate incorporation of PhIP into hair as a putative exposure biomarker for food mutagens. We have previously shown that PhIP increases intestinal tumourigenesis in C57BL/6J‐Min/+ (Multiple Intestinal Neoplasia) mice. Fur was sampled from mice exposed according to various PhIP protocols, and the amount of PhIP in the fur was quantitated by high performance liquid chromatography – mass spectrometry (HPLC‐MS). A quantitative incorporation of PhIP in the fur was demonstrated after a single subcutaneous injection of PhIP, and between one and eight PhIP exposures either via direct subcutaneous injections or through breast milk from PhIP‐injected dams. However, after higher exposures, the amount of PhIP in the fur appeared to reach saturation. After low exposures to PhIP, the number of intestinal tumours correlated with the amount of PhIP in the fur of individual mice, whereas after higher exposures, the number of tumours was relatively higher than the amounts of incorporated PhIP in the fur. Other factors, e.g. amounts of reactive PhIP metabolites formed, are also determining the number of intestinal tumours. The demonstrated quantitative incorporation of PhIP into mice fur and the correlation with number of intestinal tumours at the lower exposures, indicate that determination of PhIP in human hair may be a suitable biomarker for exposure to dietary PhIP, which is found in human hair in 10^?3 lower amounts than in these mice.     

Age‐Dependent Induction of Aberrant Crypt Fociin Rat Colon by 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine and Azoxymethane

Pups and adult rats received seven oral exposures (three time weekly) of the food mutagen PhIP (50 mg/kg), or two subcutaneous exposures (once weekly) of the experimental carcinogen azoxymethane (3.75 mg/kg). Aberrant crypt foci (ACF) were scored 8 weeks after the first exposure. In addition, lactating dams with suckling pups were orally exposed to 50 mg/kg of PhIP, three times weekly for three weeks. Direct PhIP exposure of pups induced 2.2 times more ACF than similar exposure of adult rats (2.0±0.0 versus 0.9±0.8, P<0.05). The growth of ACF, expressed as crypt multiplicity AC/ACF, was 3.5 times larger in neonatally exposed rats than in rats exposed in adulthood (8.0±7.3 versus 2.3±1.6, P<0.05). PhIP exposure via breast milk induced ACF in 3 of 25 animals. However, the difference versus controls, which had no ACF, did not reach statistical significance. Contrary to PhIP, azoxymethane induced more ACF in adult rats than in pups (2.8±1.9 versus 4.8±1.7, P<0.05). Similarly to PhIP however, azoxymethane induced 3.2 times larger ACF (AC/ACF) in pups than in adult rats (11.9±8.4 versus 3.7±1.9, P<0.001). Whereas no PhIP‐induced ACF (0/15) were observed in the lymphoid follicles, approximately 60% of the azoxymethane‐induced ACF (32/56) were located in these structures. This difference was statistically significant (P<0.001). The density of azoxymethane‐induced ACF was 80 times larger in the lymphoid follicles than in the surrounding mucosa (P<0.01). Based on the assumption that the formation of ACF with high multiplicity is predicative for the tumour development we conclude that neonatal rats are more susceptible to PhIP and azoxymethane than adult rats.     

Aryl hydrocarbon receptor‐mediated effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on glucose‐stimulated insulin secretion in mice

Epidemiological and laboratory studies suggested that exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) affects glucose homeostasis and increases the incidence of type 2 diabetes mellitus. To evaluate the effects of TCDD on insulin secretion from islets of Langerhans (islets), we designed in vivo, ex vivo and in vitro experiments. For the in vivo experiment, male C57BL/6J and aryl hydrocarbon receptor (AhR)‐null mice were injected intraperitoneally with TCDD (10 μg kg^?1 b.w.), fasted for 12 h and administered glucose 24 h post‐administration. TCDD exposure significantly decreased the plasma insulin concentration at 60 and 120 min after a glucose challenge in C57BL/6J mice but not in AhR‐null mice. In contrast, the plasma glucose concentration was not changed by TCDD exposure in both C57BL/6J and AhR‐null mice. For the ex vivo experiment, we isolated islets 24 h after TCDD administration and determined the glucose‐stimulated insulin secretion from the islets. The insulin secretion level was found to be significantly decreased by TCDD exposure at 60 min after glucose treatment. For the in vitro experiment, islets harvested from untreated C57BL/6J mice were exposed to 0.1, 1, 10 or 100 nM TCDD for 24 h and analyzed for glucose‐stimulated insulin secretion. Insulin secretion from the islets remained unchanged regardless of TCDD dose. In conclusion, TCDD exposure impaired the second phase of glucose‐stimulated secretion of insulin from the islets via the AhR signaling pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Semiquinone fraction isolated from Bacillus sp. INM‐1 protects hepatic tissues against γ‐radiation induced toxicity

Present study was focused on evaluation of a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM‐1 for its ability against γ radiation induced oxidative stress in irradiated mice. Animals were divided into four group, i.e., (i) untreated control mice; (ii) SQGD treated (50 mg/kg b. wt. i.p.) mice; (iii) irradiated (10 Gy) mice; and (iv) irradiated mice which were pre‐treated (?2 h) with SQGD (50 mg/kg b. wt. i.p.). Following treatment, liver homogenates of the treated mice were subjected to endogenous antioxidant enzymes estimation. Result indicated that SQGD pre‐treatment, significantly (P < 0.05) induced superoxide dismutase (SOD) (19.84 ± 2.18% at 72 h), catalase (CAT) (26.47 ± 3.11% at 12 h), glutathione (33.81 ± 1.99% at 24 h), and glutathione‐S‐transferase (24.40 ± 2.65% at 6 h) activities in the liver of mice as compared with untreated control. Significant (P < 0.05) induction in SOD (50.04 ± 5.59% at 12 h), CAT (62.22 ± 7.50 at 72 h), glutathione (42.92 ± 2.28% at 24 h), and glutathione‐S‐transferase (46.65 ± 3.25 at 12 h) was observed in irradiated mice which were pre‐treated with SQGD compared with only irradiated mice. Further, significant induction in ABTS⁺ radicals (directly proportional to decrease mM Trolox equivalent) was observed in liver homogenate of H₂O₂ treated mice which were found to be significantly inhibited in H₂O₂ treated mice pre‐treated with SQGD. Thus, it can be concluded that SQGD treatment neutralizes oxidative stress caused by irradiation not only by enhancing endogenous antioxidant enzymes but also by improving total antioxidant status of cellular system and thus cumulative effect of the phenomenon may contributes to radioprotection. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1471–1478, 2014.     

Panax notoginseng saponins promote hair follicle growth in mice via Wnt/β-Catenin signaling pathway

Panax notoginseng saponins (PNS), the active ingredients of the traditional Chinese medicine Panax notoginseng, have strong neuroprotective and anti-platelet aggregation effects. To investigate whether PNS can promote hair follicle growth in C57BL/6J mice, the optimal concentration of PNS was initially determined, followed by clarification of the mechanism underlying their effects. Twenty-five male C57BL/6J mice had the hair on a 2 × 3 cm² area of the dorsal skin shaved and were equally divided into five groups: control group, 5% minoxidil (MXD) group, and three PNS treatment groups [2% (10 mg/kg), 4% (20 mg/kg), and 8% (40 mg/kg) PNS]. They were then intragastrically administered the corresponding drugs for 28 days. The effects of PNS on C57BL/6J mice were analyzed by subjecting their dorsal depilated skin samples to different assessments, including hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting (WB). The group with 8% PNS exhibited the largest number of hair follicles from 14 days onwards. Compared with the control group, the number of hair follicles increased significantly in the mice treated with 8% PNS and 5% MXD, which significantly increased in a PNS-dose-dependent manner. Immunohistochemistry and immunofluorescence results revealed that treatment with 8% PNS activated the metabolism of hair follicle cells, with them showing higher rates of proliferation and apoptosis than those in the normal group. In qRT-PCR and WB analysis, the expression of β-catenin, Wnt10b, and LEF1 was upregulated in the PNS and MDX groups compared with that in the control group. Examination of the WB bands revealed that the greatest inhibitory effect of Wnt5a occurred in mice in the 8% PNS group. PNS may promote the growth of hair follicles in mice, with 8% PNS demonstrating the strongest effect. The mechanism behind this may be related to the Wnt/β-catenin signaling pathway.     

Genetic polymorphisms in metabolism of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b] pyridine

Heterocyclic amines (HCAs) are naturally produced during common cooking processes for meats and fish. HCAs are metabolized by various enzymes, including cytochromes P450, N‐acetyl transferases, and sulfotransferases, and their bioactivated metabolites are considered to bind to DNA or protein to show carcinogenic effects. More than 20 HCAs have been identified, of which 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is classified as ‘reasonably anticipated to be a human carcinogen’ to develop cancers in breast, colon and prostate. The purpose of this study was to evaluate human exposure levels of PhIP and to understand the role of genetic polymorphisms of enzymes on PhIP metabolism. Urine samples were collected from subjects (n = 100) before 3‐day meat‐restricted diets. Subjects consumed grilled chicken, and their blood and urine were collected before and after the administration of the chickens to investigate genetic polymorphisms and PhIP levels. The mean PhIP levels were 4.22 ± 0.12, 0.61 ± 0.19 and 22.64 ± 1.00 pg ml^?1 in urine under normal conditions and before and after chicken administration, respectively. Among 21 Single‐nucleotide polymorphisms (SNP) of CYP1A1, CYP1A2, NATs and UGTs investigated in this study, genotypic groups of CYP1A1/T6235C (MSP I) and CYP1A2/?2467delT showed significant differences in PhIP excretion (P < 0.05). These results suggest that genetic polymorphisms might affect PhIP metabolism, which could improve understanding of populations subject to PhIP‐derived health risk. Copyright © 2011 John Wiley & Sons, Ltd.     

The effects of cocaine on operant responding for food in several strains of mice

 The availability of numerous genetically homogenous mouse strains permits the analysis of genetic influences on behavior and also behavioral sensitivity (responsivity) to drugs of abuse. The current study was conducted to characterize discriminated operant responding for food in four inbred strains (Balb/cByJ, DBA/2J, C57BL/6J, SJL/J), an F1 Hybrid (C57BL/6×SJL), and one outbred strain (CD1) of mouse. The effect of cocaine on this operant behavior was also examined. Initially, all animals were trained to nosepoke for food on a continuous reinforcement schedule. The minimum response requirement for reinforcement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. All strains increased operant responding as the schedule of reinforcement was raised. However, significant differences in response rate and discrimination learning were observed among the various strains of mice. Cocaine administration reduced operant responding for food in Balb/cByJ, C57BL/6J, C57BL/6×SJL/J and CD1 mice at a dose of 15.0 mg/kg, whereas higher doses were required in DBA/2J mice (30.0 mg/kg) and SJL/J mice (56.0 mg/kg). These results suggest that operant performance and the effect of cocaine on this behavior is differentially influenced by genetic make-up. Received: 20 December 1996 / Final version: 4 April 1997     

The flavonolignan‐silymarin protects enzymatic,hematological, and immune system against γ‐radiation‐induced toxicity

The main focus of this study is evaluation of radioprotective efficacy of silymarin, a flavonolignan, against γ‐radiation‐induced damage to hematological, vital organs (liver and intestine), and immune system. Survival studies revealed that silymarin (administered orally for 3 days) provided maximum protection (67%) at 70 mg/kg body weight (b.wt.) against lethal 9 Gy γ‐irradiation (dose reduction factor = 1.27). The study revealed significant (p < 0.05) changes in levels of catalase (12.57 ± 2.58 to 30.24 ± 4.89 units), glutathione peroxidase (6.23 ± 2.95 to 13.26 ± 1.36 µg of reduced glutathione consumed/min/mg protein), glutathione reductase (0.25 ± 5.6 to 11.65 ± 2.83 pM NADPH consumed/min/mg protein), and superoxide dismutase (11.74 ± 0.2 to 16.09 ± 3.47 SOD U/mg of protein) activity at 30th day. Silymarin pretreated irradiated group exhibited increased proliferation in erythrocyte count (1.76 ± 0.41 × 10⁶ to 9.25 ± 0.24 × 10⁶), hemoglobin (2.15 ± 0.48g/dL to 14.77 ± 0.25g/dL), hematocrit (4.55 ± 0.24% to 37.22 ± 0.21%), and total leucocyte count (1.4 ± 0.15 × 10⁶ to 8.31 ± 0.47 × 10⁶) as compared with radiation control group on 15th day. An increase in CD4:CD8 ratio was witnessed (0.2–1%) at 30th day time interval using flow cytometry. Silymarin also countered radiation‐induced decrease (p < 0.05) in regulatory T‐cells (T_regs) (11.23% in radiation group at 7th day versus 0.1% in pretreated silymarin irradiated group at 15th day). The results of this study indicate that flavonolignan‐silymarin protects enzymatic, hematological, and immune system against γ‐radiation‐induced toxicity and might prove useful in management of nuclear and radiological emergencies. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 641–654, 2016.     

Susceptibility of metallothionein-null mice to paraquat

Using transgenic mice in which metallothionein (MT)-I and MT-II genes, we have studied a putative role of MT as a free radical scavenger against paraquat, a free radical generator. Male mice were injected s.c. with paraquat (PQ) at a single dose of 40 or 60 mg/kg of body weight (b.w.). Two of the six MT-null mice died within 16 h at the dose of 60 mg PQ/kg. b. w. PQ administration increased hepatic MT concentration in the normal mice (C57BL/6J), but not in the MT-null mice. The lipid peroxidation (LP) determined by thiobarbituric acid-reactive substance formation was increased by PQ in the liver of normal and MT-null mice, and the enhanced level was greater in the MT-null mice than in the C57BL/6J mice. Administration of PQ significantly increased blood urea nitrogen only in the MT-null mice, indicating renal damage. Without paraquat administration, the hepatic concentration of non-protein sulphydryl compounds was less in the MT-null mice than in the C57BL/6J mice, and the basal level of LP was higher in the MT-null mice than in the C57BL/6J mice. The present results support the notion that MT plays an antioxidative role against paraquat insult under physiological conditions.     

Return of the lysergamides. Part I: Analytical and behavioural characterization of 1‐propionyl‐d‐lysergic acid diethylamide (1P‐LSD)

1‐Propionyl‐d‐lysergic acid diethylamide hemitartrate (1P‐LSD) has become available as a ‘research chemical’ in the form of blotters and powdered material. This non‐controlled derivative of d‐lysergic acid diethylamide (LSD) has previously not been described in the published literature despite being closely related to 1‐acetyl‐LSD (ALD‐52), which was developed in the 1950s. This study describes the characterization of 1P‐LSD in comparison with LSD using various chromatographic and mass spectrometric methods, infrared and nuclear magnetic resonance spectroscopy. An important feature common to LSD and other serotonergic hallucinogens is that they produce 5‐HT_2A‐receptor activation and induce the head‐twitch response (HTR) in rats and mice. In order to assess whether 1P‐LSD displays LSD‐like properties and activates the 5‐HT_2A receptor, male C57BL/6 J mice were injected with vehicle (saline) or 1P‐LSD (0.025–0.8 mg/kg, IP) and HTR assessed for 30 min using magnetometer coil recordings. It was found that 1P‐LSD produced a dose‐dependent increase in HTR counts, and that it had ~38% (ED₅₀ = 349.6 nmol/kg) of the potency of LSD (ED₅₀ = 132.8 nmol/kg). Furthermore, HTR was abolished when 1P‐LSD administration followed pretreatment with the selective 5‐HT_2A receptor antagonist M100907 (0.1 mg/kg, SC), which was consistent with the concept that the behavioural response was mediated by activation of the 5‐HT_2A receptor. These results indicate that 1P‐LSD produces LSD‐like effects in mice, consistent with its classification as a serotonergic hallucinogen. Nevertheless, the extent to which 1P‐LSD might show psychoactive effects in humans similar to LSD remains to be investigated. Copyright © 2015 John Wiley & Sons, Ltd.     

4‐Phenyl butyric acid does not generally reduce glucose levels in rodent models of diabetes

1. Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of diabetes. The aim of the present study was to investigate the effect of 4‐phenyl butyric acid (PBA), a novel chemical chaperone reducing ER stress, on serum glucose level in different strains of normal and diabetic rodents. 2. 4‐Phenyl butyric acid (1 g/kg per day, i.g.) was administered to ob/ob Type 2 diabetic mice, alloxan‐induced Type 1 diabetic mice and hydrocortisone (HC)‐induced Type 2 diabetic mice as well as normal C57BL/6J mice and Kumming mice for 14 days to evaluate its effect on serum glucose levels. In addition, mice were treated simultaneously with PBA (1 g/kg per day) and HC for 9 days to determine its preventive effect against the development of insulin resistance. PBA (0.7 and 1.4 g/kg per day) was administered to non‐obese Type 2 diabetic Goto‐Kakizaki (GK) and normal Wistar‐Kyoto (WKY) rats for 14 and 7 days, respectively, to determine its effects on serum glucose levels. 3. 4‐Phenyl butyric acid significantly reduced serum glucose levels in obese Type 2 diabetic ob/ob mice. Normoglycaemia was obtained in ob/ob mice after 4 days of PBA treatment and was maintained for up to 14 days treatment. 4. 4‐Phenyl butyric acid had no glucose‐lowering effect in alloxan‐induced Type 1 diabetic mice, HC‐induced Type 2 diabetic mice and non‐obese Type 2 diabetic GK rats. In addition, it had no beneficial effects on insulin resistance in HC‐treated mice. 5. 4‐Phenyl butyric acid did not affect normal serum glucose levels in C57BL/6 J mice, Kunming mice or WKY rats. 6. In conclusion, PBA does not generally reduce glucose levels in rodent models of diabetes, although it can normalize glucose levels in ob/ob diabetic mice, indicating that restoration of ER function as diabetes therapy is limited to conditions under which ER stress is involved in the high glucose levels.     

Drug-induced potentiation of prepulse inhibition of acoustic startle reflex in mice: a model for detecting antipsychotic activity?

RATIONALE: Schizophrenic patients typically have impaired startle habituation (SH) and prepulse inhibition of the startle reflex (PPI). PPI can be disrupted in rats by psychomimetics, and drug-induced reversal of this deficit is considered to predict potential antipsychotic properties. Certain strains of mice, such as C57BL/6J, naturally display poor PPI. OBJECTIVE: To test whether mice spontaneously showing low levels of PPI might prove a useful tool for detecting novel antipsychotics. METHODS: PPI and SH were evaluated in four strains of mice: BALB/cByJ, MORO, 129/SvEv and C57BL/6J. The effects of antipsychotic [haloperidol (1, 3 and 6 mg/kg), clozapine (0.3, 1, 3 and 30 mg/kg) and risperidone (0.1, 0.3 and 1 mg/kg)] and non-antipsychotic [diazepam (3, 10 and 30 mg/kg), buspirone (1, 3 and 10 mg/kg), desipramine (3, 10 and 30 mg/kg), morphine (3, 10 and 30 mg/kg) and scopolamine (0.3, 1 and 3 mg/kg)] drug treatments were studied on PPI. RESULTS: Haloperidol (6 mg/kg), clozapine (3 and 30 mg/kg), and risperidone (1 mg/kg) all significantly enhanced PPI in C57BL/6J. All non-antipsychotics failed to improve PPI in this strain, except diazepam. Facilitation of PPI was also obtained in the other strains; however, clear interstrain differences were observed depending on the class of antipsychotic used and on the level of prepulse intensity. CONCLUSION: Antipsychotic-induced facilitation of PPI is clearly detected in mice naturally exhibiting poor levels of sensorimotor gating (e.g., C57BL/6J), but is also observed in other strains of mice. The use of this procedure as a potential screening test for detecting novel antipsychotic medications is discussed.     

Return of the lysergamides. Part II: Analytical and behavioural characterization of N6‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S,4’S)‐lysergic acid 2,4‐dimethylazetidide (LSZ)

Lysergic acid N ,N ‐diethylamide (LSD) is perhaps one of the most intriguing psychoactive substances known and numerous analogs have been explored to varying extents in previous decades. In 2013, N ⁶‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S ,4’S )‐lysergic acid 2,4‐dimethylazetidide (LSZ) appeared on the ‘research chemicals’/new psychoactive substances (NPS) market in both powdered and blotter form. This study reports the analytical characterization of powdered AL‐LAD and LSZ tartrate samples and their semi‐quantitative determination on blotter paper. Included in this study was the use of nuclear magnetic resonance (NMR) spectroscopy, gas chromatography‐mass spectrometry (GC‐MS), low and high mass accuracy electrospray MS(/MS), high performance liquid chromatography diode array detection and GC solid‐state infrared analysis. One feature shared by serotonergic psychedelics, such as LSD, is the ability to mediate behavioural responses via activation of 5‐HT_2A receptors. Both AL‐LAD and LSZ displayed LSD‐like responses in male C57BL/6 J mice when employing the head‐twitch response (HTR) assay. AL‐LAD and LSZ produced nearly identical inverted‐U‐shaped dose‐dependent effects, with the maximal responses occurring at 200 µg/kg. Analysis of the dose responses by nonlinear regression confirmed that LSZ (ED₅₀ = 114.2 nmol/kg) was equipotent to LSD (ED₅₀ = 132.8 nmol/kg) in mice, whereas AL‐LAD was slightly less potent (ED₅₀ = 174.9 nmol/kg). The extent to which a comparison in potency can be translated directly to humans requires further investigation. Chemical and pharmacological data obtained from NPS may assist research communities that are interested in various aspects related to substance use and forensic identification. Copyright © 2016 John Wiley & Sons, Ltd.     

Developmental changes in the geometry,function and responsiveness of the mouse heart to β‐adrenergic stimulation as determined by high‐resolution echocardiography

1. The mouse is the preferred species for gene targeting as a tool for research into the heart and heart development. The developmental features of the geometry and function of the heart in young mice are not well defined and cardiac functional responses following stimulation of β‐adrenoceptors have not been investigated. 2. Using the VisualSonic (Toronto, ON, Canada) high‐resolution ultrasound system, we investigated male C57BL/6 mice at 0.5–18 weeks of age. Echocardiography was performed at baseline and repeated after administration of the β‐adrenoceptor agonist isoproterenol (4 μg/kg). 3. The geometry of the left ventricle became mature 2 weeks after birth. A significant decline in left ventricular contractile function occurred at 2–3 weeks of age. 4. Inotropic and chronotropic responses to isoproterenol were significantly weaker in mice at a weaning age < 2 weeks compared with adult mice (heart rate increment 3 ± 3% vs 32 ± 4%, respectively; fractional shortening increment 19 ± 5% vs 78 ± 8%, respectively; P < 0.001). 5. In conclusion, significant changes occur in mice from birth until weaning with respect to the topography, function and β‐adrenoceptor responsiveness of the heart. The results of the present study provide a reference point for future studies in genotyping cardiac function during the postnatal phase in genetically engineered mice.     

Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice

 The dose-response relationship of cytochrome P4501b1 (Cyp1b1) and Cyp1a1 induction in livers of TCDD-treated female C57BL/6J and DBA/2J mice are described. The animals were treated i.p. with 0.001, 0.01, 0.1, 1, 10 and 50 μg TCDD/kg for 24 h, and Cyp1b1 and Cyp1a1 mRNA expression was analyzed by RT-PCR. In the livers of both mouse strains, the Cyp1b1 and Cyp1a1 mRNA content was increased after TCDD exposure in a dose-dependent manner. These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice, although Cyp1a1 was more responsive to TCDD than Cyp1b1 in both strains. The calculated ED₅₀ values for Cyp1b1 and Cyp1a1 induction in livers of TCDD-treated C57BL/6J mice were 1.3 and 0.08 μg TCDD/kg, respectively. The corresponding values for half-maximal induction response in livers of DBA/2J mice were 3.4 μg TCDD/kg for Cyp1b1 and 1.5 μg TCDD/kg for Cyp1a1. These results show that Cyp1b1 mRNA expression is less inducible by TCDD than Cyp1a1. Both genes are highly inducible in TCDD-responsive C57BL/6J mice expressing the high affinity arylhydrocarbon receptor (Ah receptor), suggesting that Cyp1b1, like Cyp1a1, is a potential Ah receptor-regulated gene. Received: 8 December 1995/Accepted: 6 February 1996     

Increased methylmercury toxicity related to obesity in diabetic KK‐Ay mice

We examined the toxic effects of methylmercury (MeHg) in KK‐Ay type 2 diabetic mice to clarify how metabolic changes associated with type 2 diabetes mellitus affect MeHg toxicity. MeHg (5 mg Hg kg ^–1day^–1 p.o.) was given to 4‐week‐old male KK‐Ay and C57BL/6J (BL/6) mice three times per week for 6 weeks. Average body weights (BW) of vehicle‐treated BL/6 and KK‐Ay mice were 16.3 and 16.4 g respectively on the first day, and 24.8 and 42.3 g respectively on the last day of the experiment. MeHg‐treated KK‐Ay mice began to lose weight about 5 weeks after MeHg administration. Six of seven MeHg‐treated KK‐Ay mice showed hind‐limb clasping in the final stage of the experiment. The mean blood mercury level of MeHg‐treated KK‐Ay mice reached a maximum of 9.8 µg ml^–1, whereas that of the MeHg‐treated BL/6 mice was 2.8 µg ml^–1 after 10 days of treatment. The average total mercury concentrations in the cerebrum and epididymal fat pad were 7.4 and 0.57 µg g^–1, respectively, for BL/6 mice and 27 and 1.6 µg g^–1, respectively, for KK‐Ay mice. In MeHg‐treated KK‐Ay mice with neurological symptoms, CD204‐positive macrophages were observed in the brain, kidney and spleen, indicating CD204 could be a marker for injured tissues. BW loss and significant pathological changes were not observed in other groups of mice. These results indicate that body fat gain in type 2 diabetes mellitus and low mercury accumulation in adipose tissue increased MeHg concentrations in organs and enhanced toxicity in KK‐Ay mice at the same dose of MeHg per BW. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential susceptibility to acetaminophen-induced liver injury in sub-strains of C57BL/6 mice: 6N versus 6J

Mouse models of acetaminophen (APAP) hepatotoxicity are considered relevant for the human pathophysiology. The C57BL/6 strain is most popular because it is the background strain of gene knock-out mice. However, conflicting results in the literature may have been caused by sub-strain mismatches, e.g. C57BL/6J and C57BL/6N. This study was initiated to determine the mechanism behind the sub-strain susceptibility to APAP toxicity. C57BL/6N and C57BL/6J mice were dosed with 200 mg/kg APAP and sacrificed at different time points. C57BL/6N mice developed significantly more liver injury as measured by plasma ALT activities and histology. Although there was no difference in glutathione depletion or cytochrome P450 activity between groups, C57BL/6N had a higher glutathione disulfide-to-glutathione ratio and more APAP protein adducts. C57BL/6N showed more mitochondrial translocation of phospho-JNK and BAX, and more release of mitochondrial intermembrane proteins apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), which caused more DNA fragmentation. The increased mitochondrial dysfunction was confirmed in vitro as C57BL/6N hepatocytes had a more precipitous drop in JC-1 fluorescence after APAP exposure. Conclusion: C57BL/6N mice are more susceptible to APAP-induced hepatotoxicity, likely due to increased formation of APAP-protein adducts and a subsequent enhancement of mitochondrial dysfunction associated with aggravated nuclear DNA fragmentation.     

Demonstration of Benzo(a)pyrene-Induced DNA Damage in Mice by Alkaline Single Cell Gel Electrophoresis: Evidence for Strand Breaks in Liver but Not in Lymphocytes and Bone Marrow

Abstract: Alkaline single cell gel electrophoresis (also known as the‘comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3×100 mg benzo(a)pyrene/kg b.wt.). No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3×150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.