Possibility of selection against mtDNA mutations in tumors.

Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutations in mitochondrial DNA (mtDNA), not observed in adjacent intact tissues. These findings were interpreted as indicating the involvement of mtDNA mutations in tumorigenesis. Such comparisons, however, ignore an important confounding factor: the monoclonal origin of tumors as opposed to the highly polyclonal nature of normal tissues. Analysis of recently published data on the incidence of somatic mutations in nontumor monoclonal cells suggests that, contrary to the prevailing view, the process of tumorigenesis may be accompanied by active selection against detrimental mtDNA mutations.

Contribution of de novo point mutations to the overall mutational burden in mitochondrial DNA of adult rats.

This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3+/-1.9 vs. 3.8+/-4.5 x 10(-4) mutations/bp, mean+/-SD), which failed to reach statistical significance (p=0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3+/-0.4 and 0.5+/-0.6 x 10(-4), respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squared=9.7, p < or = 0.01) and in older animals (Chi-squared=10.9, p < or = 0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated.

Influence of dietary antioxidants on the mutagenicity of 7,12-dimethylbenz[a]anthracene and bleomycin in female rats.

Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test.

A significant fraction of human cancers are thought to have a genetic component and several lines of evidence suggest that deficiencies in DNA repair may be a contributing factor. Little is known, however, about the frequency and distribution of variants of DNA repair genes in the general human population. The protein truncation test (PTT) was used to screen 136 healthy volunteers for protein-truncating variants of 10 DNA repair genes: APE, CDK7, ERCC1, WAF1, HOGG1, MGMT, POLB, UNG, HAAG, and CCNH. This sample consisted of 41males (30%) and 95 females (70%) with an average age of 25.3 years, ranging from 17 to 60 years of age. No truncating mutations were found in the 10 genes examined in any of the subjects. The 95% confidence interval for a proportion of 0 over the 272 alleles examined per locus is 0-0.01. The calculated frequency of truncating mutations in each of these genes, among the general population, is thus less than 1%. Among the 10 genes tested in 136 people, a single sample had no PCR product for HAAG, even though PCR products were obtained on all other genes. Total RNA dot hybridization confirmed the presence of HAAG mRNA transcripts in this sample. Despite identification of this single DNA repair variant, these results indicate a low frequency of truncating mutations in DNA repair genes in the general population.

Russian mutational spectrum differs from that of their Western counterparts.

It has been previously noted that the hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutant frequency of Russian subjects is significantly higher than age-matched Western counterparts. To further explore this difference, approximately 100 mutants collected from Russian twins reported in a previous study have been sequenced and compared to an aged-matched Western mutant dataset. The mutational spectrum of the Russian subjects was significantly different (Adams and Skopek Monte Carlo test, P = 0. 004). Curiously, this younger Russian spectrum resembles that recovered from older individuals in the West. Specifically, A:T-->C:G transversions are significantly over-represented (Fisher's Exact test, P = 0.003) in the twin spectrum as compared to the young (age

Study on genotoxic effects of the space environment: a comparison between experienced cosmonauts and unexposed Russian twins.

The molecular analysis of T-lymphocytes from experienced cosmonauts and seven pairs of unexposed twins was performed [M. Khaidakov, D. Young, H. Erfle, A. Mortimer, Y. Voronkov, B.W. Glickman, Molecular analysis of mutations in T-lymphocytes from experienced soviet cosmonauts, Environ. Mol. Mutagen, 30 (1997) 21-30; J. Curry, G. Bebb, J. Moffat, D. Young, M. Khaidakov, A. Mortimer, B.W. Glickman, Similar mutant frequencies observed between monozygotic twins, Human Mutation, 9 (1997) 445-451]. Hprt mutant frequencies (MF) in both datasets were considerably higher (38.0+/-14.6x10(-6) in cosmonauts, and 18.5+/-8.9x10(-6) in twins) than in the background Western control (8-12x10(-6)), [A.D. Tates, F.J. van Dam, H. van Mossel, H. Shoemaker, J.C.P. Thijssen, V.M. Woldring, A.H. Zwinderman, A.T. Natarajan, Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations, Mutation Res., 253 (1991) 199-213; R.F. Branda, L.M. Sullivan, J.P. O'Neill, M.T. Falta, J.A. Nicklas, B. Hirsch, P.M. Vacek, R.J. Albertini, Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations, Mutation Res., 285 (1993) 267-279]. The distribution of mutations by class in the twin dataset was essentially similar to the background Western control, whereas cosmonaut samples demonstrated a significant excess of splice errors and complex mutations. The distribution of base substitutions showed similar trends in both the cosmonaut and twin samples, which are quite distinct compared to those seen in the Western control. The differences observed between cosmonaut and twin samples (a 2-fold higher MF and an excess of complex mutations in cosmonaut mutational spectra) could be an indication of possible effects of the space environment. However, these changes could also be age-related because the twin group was, on average, 17 years younger. Moreover, very similar patterns of base substitution distribution in both datasets suggest the involvement of certain region-specific factors reflected in mutational spectra. In order to discriminate between occupation and region-specific factors contributing to mutagenesis, an additional study involving trainees and cosmonauts with recent long-term flight experience is required.

Molecular analysis of mutations in T-lymphocytes from experienced Soviet cosmonauts.

Somatic mutation in five cosmonauts who have completed spaceflights of 7 to 365 days was analyzed using the clonal HPRT assay. The doses received in space by the cosmonauts ranged from 4 to 127 mGy. hprt mutant frequencies were 2.4-5.0-fold higher than age-corrected values established for healthy, unexposed subjects in western countries [Tates et al. (1991): Mutat Res 253: 199-213; Branda et al. (1993): Mutat Res 285: 267-279] and 2- to 3-fold higher than those determined for unexposed individuals residing in Russia [Jones et al. (1995): Mutat Res 338: 129-139]. A total of 107 collected mutant clones were analyzed by multiplex PCR. No excess of deletions was detected and their frequency did not correlate with either accumulated dose or the age of the cosmonauts. In 62 mutants cDNA was isolated by RT-PCR and sequenced. Those with splicing errors, as well as the mutants that did not produce cDNA, were further analyzed by the sequencing of exon(s)-containing fragments amplified from genomic DNA. The mutational spectrum recovered from the cosmonauts differed substantially from that of unexposed healthy subjects (P = 0.042), and exhibited an increased incidence of splicing errors, frameshifts, and complex mutations. Higher frequencies of contribution of AT-->GC transitions and GC-->TA transversions were also observed. The increased mutant frequencies and observed shifts in mutational spectra likely indicate a combination of potential influences, including environment, lifestyle, and occupational exposures. Further elucidation of these potential influences will require a more extensive study involving the general population sharing similar environment, cosmonauts in training and cosmonauts participating in space flights.

Similar mutant frequencies observed between pairs of monozygotic twins.

The relative contribution of both genetic and environmental factors to spontaneous mutation frequency in humans is unknown. We have investigated the contribution of genetic factors to this phenomenon by determining the in vivo mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in circulating T-lymphocytes obtained from pairs of monozygotic twins. hprt mutant frequencies were determined three times over fourteen days in six sets of monozygotic male twins (mean age 30) taking part in a Russian Space Program inclined bed rest experiment. Blood samples were obtained prior to, during, and immediately following the experiment. Mononuclear cells were separated, frozen, and flown to Canada for analysis using the hprt T-lymphocyte clonal assay. There is no evidence within this data set to demonstrate that the period of inclined bed rest to simulate the effects of weightlessness had any effect on the observed mutant frequency. However, the average mutant frequency for the six sets of Russian twins was found to be three times higher than that of Western counterparts. More surprisingly, the spontaneous mutant frequency of monozygotic twins was found to be much more similar within pairs than between pairs of twins. These data suggest that the contribution of genetics in the determination of mutation frequency is substantial. However, whether high concordance within twin pairs reflects shared environmental experience as well as common genetic factors is not entirely clear. More data will be required to distinguish genetic from environmental factors and to determine the degree to which mutant frequency is genetically determined.

Possible factors leading to a misjudgement of mutant frequencies in HPRT assay.

Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay in in vitro study were analysed. In 12 separate reconstruction experiments with independent pairs of wild type (WT) and mutant (HPRT-) clones, the CE of WT cells (Group 1) and the recovery of mutant cells in absence (Group 2), as well as in the presence of non-irradiated (Group 3), or irradiated (Group 4) WT cells (10(4) cells/well) was determined. The plating of mutant cells with irradiated WT cells improved their CEs by almost 30%. In contrast, the presence of non-irradiated WT cells led to a slight decline (10%) in CE of mutant cells, resulting in a significant difference between groups (p = 0.0083). The extent of decline in survival of mutant cells in the presence of non-irradiated WT cells negatively correlated (r = 0.3496, p < 0.05) with the initial CE of WT cells. The data suggest that the presence of WT cells in the selection plates may suppress the recovery of mutants in HPRT assay, and this negative effect is stronger in samples with high CE. These findings indicate a possible source for a serious underestimation of mutant frequencies (3-fold in the range of CEs from 10% to 60%) in the HPRT assay and may be useful for the interpretation of results from studies on exposure to mutagens in humans.

Mutational specificity and cancer chemoprevention.

Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment. The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context. As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation. Conversely, mutational spectra should also provide insights into the identity of the mutagen. Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations. With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial. Both in vitro and in vivo data has been obtained following exposure to a variety of agents. In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations. The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed.

[The duration of the aftereffects of UV-irradiation of man in conditions of UV-insufficiency]. / O dlitel'nosti posledeistviia UF-oblucheniia cheloveka v usloviiakh UF-nedostatochnosti.

The purpose of the present investigation was to study the aftereffect of UV-B-irradiation applied during 10 or 20 sessions. The biological effect and aftereffect duration of UV-B-irradiation of the upper body of 9 healthy volunteers (residents of the city of Moscow and its suburbs) were evaluated in autumn and winter. During UV-irradiation in the range 220-280, 280-320, or 320-400 nm the flux density was 0.035, 1.75, or 0.65 W/m2, respectively. Group 1 subjects (5 men) were exposed to UV-B-irradiation 10 times (once with 0.5, 0.6, 0.75, 0.9, 1.1, 1.35, 1.65 MED and three times with 2.0 MED) and Group 2 subjects (4 men) were exposed to UV-B-irradiation 20 times (twice with 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 MED and eight times with 2 MED). The sessions were arranged every other day. The exposure produced a beneficial effect on the human body: skin sensitivity decreased, resistance of skin capillaries increased, Ca metabolism normalized, blood 25 (OH) D grew. The trend of responses was similar after 10 and 20 sessions; however, 20 sessions caused a longer-term aftereffect. In relation to space programs, it is concluded that UV-irradiation can be applied on the ground before short-term flights (less than 4 months) and in space during longer-term flights (greater than 4 months).

[Effect of active metabolites of vitamin D 3 on the status of rat bone tissue in various experimental models of hypokinesia]. / Vliianie aktivnykh metabolitov vitamina D 3 na sostoianie kostnoi tkani krys pri razlichnykh éksperimenta'lnykh modelakh gipokinezii.

Rats were for 6 weeks either kept in small cages or suspended. The caged rats showed hypocalcemia and lowered active transport of calcium in the intestine and no changes of PTH in blood. Femoral bone measurements in these rats revealed reduced density and content of calcium and phosphorus in proximal epiphyses, slight increase of these parameters in diaphyses and lack of changes in distal epiphyses. The suspended rats exhibited normocalcemia, noticeable but insignificant increase of PTH and calcium absorption as well as decreased density and content of calcium and phosphorus in distal epiphyses and their slight increase in diaphyses. Administration of active vitamin D3 metabolites led to an increase of bone mineral density and content only in those femur compartments where hypokinesia-induced changes were seen. It is concluded that during hypokinesia bone disorders are predominantly produced by local factors that may increase bone sensitivity to systemic influences.

[Quantitative study of the osteoblasts and osteoclasts in the bones of rats during the simulation of weightlessness]. / Kolichestvennoe issledovanie osteoblastov i osteoklastov v kostiakh krys pri modelirovanii nevesomosti.

Tibia and vertebrae of rats exposed to hypokinesia or head-down suspension were investigated by quantitative histomorphometry. It was found that 35- and 60-day hypokinesia as well as 35-day suspension caused osteoporosis in the tibial and vertebral spongiosa. The development of osteoporosis was accompanied by a significant reduction in the number of osteoblasts in the primary spongiosa of tibia and vertebrae whereas no noticeable changes in osteoclasts were observed either in hypokinetic or suspended rats. The only exception was lumbar vertebrae in which the amount of osteoclasts decreased as a result of 60-day hypokinesia. It is assumed that the reduction in the number and activity of osteoblasts plays the major part in the development of osteoporosis during hypokinesia and suspension.

[Comparative study of the toxicity of 1 alpha-hydroxyvitamin D3 and 24,25 (RS)-dihydroxyvitamin D3 in rats]. / Sravnitel'noe izuchenie toksichnosti 1 al'fa-oksivitimina D3 i 24,25 (RS)-dioksivitamina D3 u krys.

The toxic effects of 1 alpha (OH)D3 and 24,25 (OH)2D3 administered in doses of 0.25, 2.5 and 25 micrograms per animal a day were compared in rats weighing initially 230-260 g and fed an artificial diet containing 0.65 and 0.50% of Ca and P, respectively. After 5 days of administering different doses of 1 alpha (OH) D3 hypercalcemia and hyperphosphatemia developed whatever the dose, the animals' weight and density of the osseous tissue dropped starting with a dose of 2.5 micrograms, together with a high death rate and Ca accumulation by soft tissues at a dose of 25 micrograms per animal. Unlike 1 alpha (OH)D3, 24,25 (OH)2D3 did not exert any hypercalcemic or hyperphosphatemic action when given in a high dose (25 micrograms). On the contrary, it promote the decrease of the Ca and P blood levels. 24,25 (OH)2D3 did not bring about Ca accumulation by the organs or reduction of the osseous tissue density whatever the dose applied. In addition, the metabolite administered in a dose of 25 micrograms arrested the animals' growth. Thus, when given in comparable doses (the physiologic requirement of 1 alpha (OH)D3 and 24,25 (OH)2D3 for rats are 0.025 and 0.25 micrograms/day, respectively), 24,25 (OH)2D3 was at least one order of magnitude less active as regards its capacity to increase the Ca and P blood levels and to resorb the osseous tissue. The data obtained and the inhibitory effect on the growth of the 100-fold dose of 24,25 (OH)2D3 point to the feasibility of the short-term use of the metabolite in doses that do not exceed more than 10-fold the physiologic dose.