The tobacco‐specific nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) induces mitochondrial and nuclear DNA damage in Caenorhabditis elegans

The metabolites of the tobacco‐specific nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) form DNA adducts in animal models. While there are many reports of formation of nuclear DNA adducts, one report also detected NNK‐induced damage to the mitochondrial genome in rats. Using a different DNA damage detection technology, we tested whether this finding could be repeated in the nematode Caenorhabditis elegans. We treated N2 strain (wild‐type) nematodes with NNK in liquid culture, and applied quantitative PCR to analyze NNK‐induced nuclear and mitochondrial DNA (mtDNA) damage. Our results confirm that NNK causes both nuclear and mtDNA damage. However, we did not detect a difference in the level of nuclear versus mtDNA damage in C. elegans. To test whether the mtDNA damage was associated with mitochondrial dysfunction, we used a transgenic nematode strain that permits in vivo measurement of ATP levels and found lower levels of ATP in NNK‐exposed animals when compared with the unexposed controls. To test whether the lower levels of ATP could be attributed to inhibition of respiratory chain components, we investigated oxygen consumption in whole C. elegans and found reduced oxygen consumption in exposed animals when compared with the unexposed controls. Our data suggest a model in which NNK exposure causes damage to both C. elegans nuclear and mitochondrial genomes, and support the hypothesis that the mitochondrial damage is functionally important in this model. These results also represent a first step in developing this genetically tractable organism as a model for assessing NNK toxicity. Environ. Mol. Mutagen. 55:43–50, 2014. © 2013 Wiley Periodicals, Inc.     

Metabolism of hyperpolarized 13C‐acetoacetate to β‐hydroxybutyrate detects real‐time mitochondrial redox state and dysfunction in heart tissue

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter‐conversion of β‐hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3‐¹³C]acetoacetate to [1,3‐¹³C]β‐hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) ¹³C‐acetoacetate (AcAc) to ¹³C‐β‐hydroxybutyrate (β‐HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP‐AcAc to β‐HB was investigated using ¹³C magnetic resonance spectroscopy in Langendorff‐perfused rat hearts in four groups: control, global ischemic reperfusion, low‐flow ischemic, and rotenone (mitochondrial complex‐I inhibitor)‐treated hearts. We observed that more β‐HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in β‐HB, as detected by ¹³C MRS, was validated by a direct measure of tissue β‐HB by ¹H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD⁺/NADH, measured by enzyme assays of homogenized tissue, also paralleled production of β‐HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP‐AcAc to HP‐β‐HB detected by ¹³C‐MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.     

To have and to hold: Episodic memory in 3‐ and 4‐year‐old children

Episodic memory endows us with the ability to reflect on our past and plan for our future. Most theorists argue that episodic memory emerges during the preschool period and that its emergence might herald the end of childhood amnesia. Here, we show that both 3‐ and 4‐year‐old children form episodic memories, but that 3‐year‐old children fail to retain those memories following a delay (Experiments 1 and 2). In contrast, 4‐year‐old children retained episodic memories over delays of 24 hr (Experiment 1) and 1 week (Experiment 3). This marked change in the retention of episodic memories between 3 and 4 years of age suggests that it is our ability to retain, rather than to form, an episodic memory that limits our ability to recall episodes from early childhood. © 2011 Wiley Periodicals, Inc. Dev Psychobiol 55: 125–132, 2013     

Comparative DNA adduct formation and induction of colonic aberrant crypt foci in mice exposed to 2‐amino‐9H‐pyrido[2,3‐b]indole, 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline,and azoxymethane

Considerable evidence suggests that environmental factors, including diet and cigarette smoke, are involved in the pathogenesis of colon cancer. Carcinogenic nitroso compounds (NOC), such as N‐nitrosodimethylamine (NDMA), are present in tobacco and processed red meat, and NOC have been implicated in colon cancer. Azoxymethane (AOM), commonly used for experimental colon carcinogenesis, is an isomer of NDMA, and it produces the same DNA adducts as does NDMA. Heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and high‐temperature cooking of meats are also associated with an elevated risk of colon cancer. The most abundant carcinogenic HAA formed in tobacco smoke is 2‐amino‐9H‐pyrido[2,3‐b]indole (AαC), whereas 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ) is the most potent carcinogenic HAA formed during the cooking of meat and fish. However, the comparative tumor‐initiating potential of AαC, MeIQ, and AOM is unknown. In this report, we evaluate the formation of DNA adducts as a measure of genotoxicity, and the induction of colonic aberrant crypt foci (ACF) and dysplastic ACF, as an early measure of carcinogenic potency of these compounds in the colon of male A/J mice. Both AαC and AOM induced a greater number of DNA adducts than MeIQ in the liver and colon. AOM induced a greater number of ACF and dysplastic ACF than either AαC or MeIQ. Conversely, based on adduct levels, MeIQ‐DNA adducts were more potent than AαC‐ and AOM‐DNA adducts at inducing ACF. Long‐term feeding studies are required to relate levels of DNA adducts, induction of ACF, and colon cancer by these colon genotoxicants. Environ. Mol. Mutagen. 57:125–136, 2016. © 2016 Wiley Periodicals, Inc.     

DNA adducts,mutant frequencies,and mutation spectra in various organs of λlacZ mice exposed to ethylating agents

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, λlacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O⁶-ethylguanine (O⁶-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O⁶-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O⁶-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC → AT transitions, consistent with the O⁶-EtG lesion, and 28% TA → AT transversions, expected from O²-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC → AT mutations and 22% were TA → AT mutations. This suggests that in bone marrow O⁶-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O⁶-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O⁶- and N7-adducts were present. Environ. Mol. Mutagen. 31:18–31, 1998. © 1998 Wiley-Liss, Inc.     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.