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.epigenetic | genetic risk

Recent data show that the effects of ionising radiation are not restricted to the directly exposed parental germ cells, but can also manifest in their non-exposed offspring, resulting in elevated mutation rates and cancer predisposition. The mechanisms underlying these transgenerational changes remain poorly understood. One of the most important steps in elucidating these mechanisms is to investigate the initial cellular events that trigger genomic instability. Here we have analysed the effects of paternal treatment by ethylnitrosourea, an alkylating agent which is known to form specific types of DNA adducts, on the transgenerational effects in the first-generation (F 1 ) offspring of exposed CBA/Ca and BALB/c male mice. Mutation rates at two expanded simple tandem repeat loci were significantly elevated in the F 1 germline of both strains. Pre-and post-meiotic exposures resulted in similar increases in mutation rate in the F 1 germline. Within each strain mutation rates were equally elevated in the germline of male and female F 1 offspring of the directly exposed males. The results of our study suggest that transgenerational instability is not attributed to a specific sub-set of DNA lesions, such as double strand breaks, and is most probably triggered by a stress-like response to a generalised DNA damage.2

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 PCRbased 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 singlemolecule 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.

1 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). 2 In the presence of 300 mM L-nitroarginine and 10 mM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 mM glibenclamide whereas those to NOR-1, substance P and bradykinin were una ected. 3 Hyperpolarizations produced by maximally-e ective concentrations of NOR-1 and NS1619 were identical (to 765 mV). They were signi®cantly less than those generated by either substance P or bradykinin (to approximately 780 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no e ect on responses to substance P or bradykinin. 4 Incubation of segments of intact arteries for 16 ± 22 h in bicarbonate-bu ered Krebs solution had little e ect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-bu ered Tyrode solution or Krebs containing 10 mM 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 mM 17-octadecynoic acid. 5 We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-bu ered 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 Keywords: EDHF; iberiotoxin; porcine coronary artery; gap junctions; HEPES; bradykinin; prostacyclin; substance P; nitric oxide donor; hyperpolarization Abbreviations: BK Ca , large conductance calcium-sensitive K + channel; 1-EBIO, 1-ethyl-2-benzimidazolinone; EDHF, endotheliumderived hyperpolarizing factor; HEPES, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid); IntroductionIn small arteries and arterioles it is now well established that agonists which interact with vascular endothelial cell receptors can hyperpolarize and relax the underlying smooth muscle. It has long been assumed that the endothelium is stimulated to release an endothelium-derived hyperpolarizing factor (EDHF) which di uses across the myo-endothelial space to exert its e ects by opening smooth muscle K + channels (see reviews by Edwards & Weston, 1998; Fe le tou & Vanhoutte, 1999). However, the identity of this`factor' remains unknown and in view of the dissimilar pharmacology of the EDHF responses in di erent species and blood vessels, the possibility that several endogenous substances are involved cannot be discounted.In most vascular preparations, a characteristic feature of the EDHF response is its full inhibition by charybdotoxin+apamin but only slig...