In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR‐1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium‐derived hyperpolarizing factor (EDHF). In the presence of 300 μ M L ‐nitroarginine and 10 μ M indomethacin, iloprost‐induced hyperpolarizations were partially inhibited by 10 μ M glibenclamide whereas those to NOR‐1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally‐effective concentrations of NOR‐1 and NS1619 were identical (to −65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately −80 mV) and were abolished by iberiotoxin 100 n M , a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16–22 h in bicarbonate‐buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES‐buffered Tyrode solution or Krebs containing 10 m M HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin‐induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 μ M 17‐octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo‐oxygenase inhibitors. Incubation in HEPES‐buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K + channels, activated only by bradykinin. British Journal of Pharmacology (2001) 133 , 1145–1153; doi: 10.1038/sj.bjp.0704157
It is well known that the developing embryo is especially sensitive to ionising radiation. However, to date little is known about the long-term effects of in utero exposure on mutation rates during adulthood. To evaluate the effects of in utero irradiation on mutation induction and transgenerational instability, BALB/c pregnant mice (Theiler stage 20, 12 days of gestation) were exposed to 1 Gy of acute X-rays. The in utero exposed 8-week-old males and females were mated to control partners. To evaluate the effects of in utero irradiation on mutation induction in the germline of exposed mice, all parents and offspring were profiled using two mouse-specific expanded simple tandem repeat (ESTR) probes Ms6-hm and Hm-2. The results of our study show that ESTR mutation rates in the germline of in utero irradiated male and female mice remain highly elevated during adulthood. Using single-molecule PCR, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the in utero irradiated animals. In all animals, a statistically significant ~2.8-3.7 fold increase in the mean mutation frequency was found in all tissues of the in utero irradiated animals. The results of our study show that the mutagenic effects of in utero irradiation in mice are well manifested during adulthood and therefore suggest that the susceptibility of early stages of mouse development to ionising radiation may be higher than previously thought. To analyse the effects of parental irradiation on transgenerational instability, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the first-generation offspring of in utero irradiated male and female mice. The results of our study show that in the offspring of in utero exposed males the frequency of ESTR mutation is considerably elevated across multiple tissues, whereas in the offspring of irradiated females it does not significantly differ from that in controls. A comparison with the results of our previous studies on transgenerational in stability among the offspring of BALB/c male mice irradiated during adulthood showed that that the magnitude of transgenerational effects is not affected by the stage of paternal exposure. This work has therefore established that an instability signal induced in the germline of in utero irradiated males is manifested during adulthood. The potential implications of our findings to for further understanding of the possible mechanisms of transgenerational genomic instability will be discussed.
The genetic effects of human exposure to anticancer drugs remain poorly understood. To establish whether exposure to anticancer drugs can result not only in mutation induction in the germ line of treated animals, but also in altered mutation rates in their offspring, we evaluated mutation rates in the offspring of male mice treated with three commonly used chemotherapeutic agents: cyclophosphamide, mitomycin C, and procarbazine. The doses of paternal exposure were approximately equivalent to those used clinically. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat locus Ms6-hm was established in DNA samples extracted from sperm and bone marrow of the offspring of treated males. After paternal exposure to any one of these three drugs, expanded simple tandem repeat mutation frequencies were significantly elevated in the germ line (sperm) and bone marrow of their offspring. This observed transgenerational instability was attributed to elevated mutation rates at the alleles derived from both the exposed fathers and from the nonexposed mothers, thus implying a genome-wide destabilization. Our results suggest that paternal exposure to a wide variety of mutagens can result in transgenerational instability manifesting in their offspring. Our data also raise important issues concerning delayed transgenerational effects in the children of survivors of anticancer therapy.
Hepatocellular carcinoma is one of the most frequently occurring cancers in humans. Recent human and animal studies have provided strong evidence for the effects of dietary deficiency of methyl donors on the development of liver cancer. However, the mechanisms underlying the effects of methyl-group deficiency on cancer risk are not properly understood.Male BALB/c and CBA/Ca mice were maintained for 8 weeks on a synthetic diet lacking in choline and folic acid. Using microarrays, the pattern of gene expression was evaluated in their liver, kidney, and spleen. Methyl-donor deficient diet induced profound changes in gene expression in the liver of treated animals, whereas the effects of the methyl-deficient diet on the pattern of gene expression in the kidney and spleen were negligible. Methyl-donor dietary restriction induced strain-independent upregulation of genes involved in cellular proliferation in liver.The results of our study provide a plausible explanation of why diets lacking methyl donors can induce the development of liver cancers in rodents and humans.
Abstract Understanding and estimating the genetic hazards of exposure to chemical mutagens and anticancer drugs in humans requires the development of efficient systems for monitoring germ line mutation. The suitability of a single-molecule PCR–based approach for monitoring mutation induction at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm by chemical mutagens and anticancer drugs has been validated. The frequency of ESTR mutation was evaluated in the germ line of male mice exposed to the well-characterized alkylating agent and mutagen, ethylnitrosourea, and four widely used anticancer drugs, bleomycin, cyclophosphamide, mitomycin C, and procarbazine. The dose-response of ethylnitrosourea-induced mutation was found to be very close to that previously established using a pedigree-based approach for ESTR mutation detection. Paternal exposure to the clinically relevant doses of bleomycin (15–30 mg/kg), cyclophosphamide (40–80 mg/kg), and mitomycin C (2.5–5 mg/kg) led to statistically significant, dose-dependent increases in ESTR mutation frequencies in the germ line of treated male mice. Exposure to procarbazine led to a maximal increase in mutation frequency at 50 mg/kg, with a plateau at the higher concentrations. The results of this study show that the single-molecule PCR technique provides a new and efficient experimental system for monitoring the genetic effects of anticancer drugs, capable of detecting increases in mutation rates at clinically relevant doses of exposure. In addition, this approach dramatically reduces the number of mice needed for the measurement of germ line mutation induction. [Cancer Res 2008;68(10):3630–6]
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