increase in mutation rate in these children compared to those conceived before the Chernobyl accident and external controls. However, the mutants were detected using random amplified polymorphic DNA-PCR, an unreliable technology. These mutants were not validated and had no obvious molecular basis (Jeffreys and Dubrova 2001).
There are some limited data on minisatellite mutations detected directly in sperm sampled from cancer patients who have sustained radiotherapy and/or chemotherapy (Armour and others 1999; May and others 2000; Zheng and others 2000). All of these studies used the so-called small-pool PCR approach (SP-PCR) originally developed for the analysis of spontaneous mutations at human minisatellite loci (Jeffreys and others 1994). While this method can overcome the small sample size limitations encountered in pedigree analysis, a major shortcoming of the SP-PCR approach, compared to the pedigree approach, is the very large variation in spontaneous mutation rates of individual alleles at a single locus. Although SP-PCR can be used to evaluate the mutation rate in the same male before and after mutagenic treatment, it does not allow amplification of very large minisatellite alleles (longer than 5 kb), thus restricting mutation scoring to a subset of relatively small minisatellite sizes.
In the first of these studies (Armour and others 1999), sperm DNA of two men exposed to the anticancer drugs cyclophosphamide, etoposide, and vincristine, plus 2.2 Gy of X-rays (scattered radiation from mediastinal radiotherapy), were analyzed for mutations at the MS205 locus known to have a high germline mutation rate (~0.4–0.7% per gamete). There were no significant differences in mutation frequencies in the pretherapy and posttherapy samples (11 and 16 months, respectively, in the two individuals). Mutation rates were 0.38% versus 0.47% in the former and 0.10% versus 0.11% in the latter. It should be noted, however, that in mouse experiments, cyclophosphamide is mutagenic only in postmeiotic germ cells, etoposide (a topoisomerase II inhibitor) is mutagenic only in meiotic cells, and vincristine is not mutagenic, although it is known to prevent the assembly of tubulin into spindle fibers (Witt and Bishop 1996; Russell and others 1998).
In the second study (Zheng and others 2000), sperm DNA from 10 men treated for Hodgkin’s disease (with different combinations of chemotherapeutic agents plus 2.5 Gy of abdominal X-rays) were analyzed using the MS205 locus. Nine patients treated with either vinblastine or adriamycin and bleomycin did not show any increases in mutation frequency. Vinblastine binds to tubulin and, in mice, results in aneuploidy but not chromosome breakage or mutations. Adriamycin is an intercalating agent and an inhibitor of topoisomerase-II, and in mice, this compound is toxic to germ cells but does not cause mutations (Witt and Bishop 1996). Bleomycin, a radiomimetic agent, selectively targets mouse oocytes, but no mutation induction in male germ cells has been observed. The only patient treated with procarbazine + oncovin + prednisone (for six cycles with 3–4 week intervals between cycles) showed a slight increase in mutation frequency (1.14% versus 0.79%). Procarbazine is known to be mutagenic to mouse spermatogonia.
In the work of May and colleagues (2000), sperm DNA samples from three seminoma patients who underwent orchiectomy and external beam radiotherapy were used to study induction of mutations at the B6.7 and CEB1 loci. These men received 15 fractions of acute X-irradiation, with a total testicular dose (from scattered radiation) ranging between 0.4 and 0.8 Gy. No induced mutations were found.
The most recent DD estimates consistent with the Japanese data are those of Neel and colleagues (1990). These were expressed as “end-point-specific minimal DDs” excluded by the data at specified probability levels and “most probable gametic DD” (note that all of these are for the acute radiation conditions obtained during the bombings). For example, the minimal DDs at the 95% probability level were the following: 0.05 to 0.11 Sv (F1 cancers); 0.18 to 0.29 Sv (UPO); 0.68 to 1.10 Sv (F1 mortality); 1.60 Sv (sex-chromosomal aneuploidy), and 2.27 Sv (electrophoretic mutations). When only UPO, F1 cancers, and F1 mortality were considered together, the estimated DD at the 95% probability level was 0.63 to 1.04 Sv. The comparable estimate for sex chromosomal aneuploidy and electrophoretic mutations considered together was 2.71 Sv.
The oft-quoted DD range of 1.69 to 2.23 Sv, called the “most probable gametic DD” by Neel and colleagues, was obtained by calculating overall spontaneous and induced “mutation rates” for the above-mentioned five end-points and obtaining a ratio of these two. The former was estimated by summing the five individual estimates of spontaneous rates (which yielded 0.00632 to 0.00835 per gamete) and the latter, likewise, by summing the individual rates of induction (which yielded 0.00375 per gamete per parental Sv). The ratio 0.00632-0.00835/0.00375 is the DD range which is 1.69 to 2.23 Sv. The overall DDs thus calculated were found to be between 1.69 Sv (i.e., 0.00632/0.00375) and 2.23 Sv (i.e., 0.00835/0.00375) for the acute radiation conditions during the bombings. In these estimates, the limits reflect biological uncertainties about the parameters, but do not take into account the additional error inherent in the estimation process itself, which must be relatively large (Neel and others 1990). With a dose-rate reduction factor of 2 (which was used) for chronic low-LET radiation conditions, the relevant DD becomes about 3.4 to 4.5 Sv. Note, how-