Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations.

Deletion and translocation mutations have been shown to play a significant role in the genesis of many cancers. The hprt gene located at Xq26 is a frequently used marker gene in human mutational studies. In an attempt to better understand potential mutational mechanisms involved in deletions and translocations, inverse PCR (IPCR) methods to amplify and sequence the breakpoints of hprt mutants classified as translocations and large deletions were developed. IPCR involves the digestion of DNA with a restriction enzyme, circularization of the fragments produced, and PCR amplification around the circle with primers oriented in a direction opposite to that of conventional PCR. The use of this technique allows amplification into an unknown region, in this case through the hprt breakpoint into the unknown joined sequence. Through the use of this procedure, two translocation, one inversion, and two external deletion hprt breakpoint sequences were isolated and sequenced. The isolated IPCR products range in size from 0.4 to 1.8 kb, and were amplified from circles ranging in size from 0.6 to 7.7 kb. We have shown that inverse PCR is useful to sequence translocation and large deletion mutant breakpoints in the hprt gene.

Biomarkers for assessing occupational exposures to 1,3-butadiene.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease.

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.

hprt mutant frequencies, nonpulmonary malignancies, and domestic radon exposure: "postmortem" analysis of an interesting hypothesis.

The hypothesis that exposure to domestic radon raises the risk for leukemia and other nonpulmonary cancers has been proposed and tested in a number of epidemiologic studies over the past decade. During this period, interest in this hypothesis was heightened by evidence of increased frequencies of mutations at the hypoxanthine guanine phosphoribosyl transferase (hprt) gene in persons exposed to domestic radon (Bridges BA et al. [1991]: Lancet 337:1187-1189). An extension of this study (Cole J et al. [lsqb[1996]: Radiat Res 145:61-69) and two independent studies (Albering HJ et al. [1992[: Lancet 340:739; Albering HJ et al. [1994[: Lancet 344:750-751) found that hprt mutant frequency was not correlated with domestic radon exposure, and two well-designed epidemiologic studies showed no evidence of a relation between radon exposure and leukemia in children or adults. In this report, we present additional data from a study of Colorado high school students showing no correlation between domestic radon exposure and hprt mutant frequency. We use reanalyses of previous studies of radon and hprt mutant frequency to identify problems with this assay as a biomarker for domestic radon exposure and to illustrate difficulties in interpreting the statistical data. We also show with analyses of combined data sets that there is no support for the hypothesis that domestic radon exposure elevates hprt mutant frequency. Taken together, the scientific evidence provides a useful example of the problems associated with analyzing and interpreting data that link environmental exposures, biomarkers, and diseases in epidemiologic studies.

The effect of folate deficiency on the cytotoxic and mutagenic responses to ethyl methanesulfonate in human lymphoblastoid cell lines that differ in p53 status.

Folic acid deficiency acts synergistically with alkylating agents to increase genetic damage at the HPRT locus in Chinese hamster ovary cells in vitro and in rat splenocytes in vivo. The present studies extend these observations to human cells and, in addition, investigate the role of p53 activity on mutation induction. The human lymphoblastoid cell lines TK6 and WTK1 are derived from the same parental cell line (WI-L2), but WTK1 expresses mutant p53. Treatment of folate-replete or deficient WTK1 and TK6 cells with increasing concentrations (0-50microg/ml) of ethyl methanesulfonate (EMS) resulted in significantly different HPRT mutation dose-response relationships (P<0.01), indicating that folate deficiency increased the EMS-induced mutant frequency in both cell lines, but with a greater effect in TK6 cells. Molecular analyses of 152 mutations showed that the predominant mutation (65%) in both cell types grown in the presence or absence of folic acid was a G>A transition on the non-transcribed strand. These transitions were mainly at non-CpG sites, particularly when these bases were flanked 3' by a purine or on both sides by G:C base pairs. A smaller number of G>A transitions occurred on the transcribed strand (C>T=14%), resulting in 79% total G:C>A:T transitions. There were more genomic deletions in folate-deficient (15%) as compared to replete cells (4%) of both cell types. Mutations that altered RNA splicing were common in both cell types and under both folate conditions, representing 33% of the total mutations. These studies indicate that cells expressing p53 activity exhibit a higher rate of mutation induction but are more sensitive to the toxic effects of alkylating agents than those lacking p53 activity. Folate deficiency tends to reduce toxicity but increase mutation induction after EMS treatment. The p53 gene product did not have a major influence on the molecular spectrum after treatment with EMS, while folate deficiency increased the frequency of deletions in both cell types.

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells.

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Identification of a new Lesch-Nyhan syndrome mutation (HPRTBrasil) and analysis of potentially heterozygous females.

The mutation in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene has been determined in two brothers affected with Lesch-Nyhan syndrome. Female members of the family who are at risk for being heterozygous carriers of the HPRT mutation were also studied to determine whether they carry the mutation. DNA sequencing revealed that the boys' mother is heterozygous for the mutation in her somatic cells, but that three maternal aunts are not heterozygous. Such carrier information is important for the future pregnancy plans of at-risk females. The mutation, an A-->T transversion at cDNA base 590 (590A-->T), results in an amino acid change of glutamic acid to valine at codon 197, and has not been reported previously in a Lesch-Nyhan syndrome male. This mutation is designated HPRTBrasil.

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

Development of long PCR techniques to analyze deletion mutations of the human hprt gene.

DNA primers and reaction conditions for long polymerase chain reaction amplification (PCR) of portions of the human hprt gene are presented. Use of these primers with DNA from previously defined hprt deletion mutants demonstrated production of the expected smaller size products as compared to DNA from wild type cells. These primers will be of value in the rapid analysis and subsequent sequencing of hprt deletion mutations in human cells.

Nutritional folate deficiency augments the in vivo mutagenic and lymphocytotoxic activities of alkylating agents.

To investigate the interaction of folate deficiency and alkylating agents in vivo, weanling Fischer 344 rats were maintained for 5 weeks on a folate replete, moderately folate deficient, or a severely folate deficient diet. Mutant frequencies at the HPRT locus in splenic lymphocytes were 1.2+/-0.6, 1.9+/-1.1, and 6.4+/-4.0 x 10(-6), respectively (P < 0.01). N-nitroso-N-ethylurea (ENU), 100 mg/kg body weight, was much more mutagenic with progressive folate deficiency (5.0+/-2.4 vs. 16.2+/-7.3 vs. 39.2+/-21.0 x 10(-6)), suggesting a synergistic interaction (P << 0.01). Neither moderate nor severe folate deficiency significantly enhanced the mutagenic effects of cyclophosphamide, 50 mg/kg body weight (18.0+/-7.9 vs. 6.0+/-2.8 vs. 28.5+/-28.2 x 10(-6)). The number of cloning cells/ spleen were reduced 68% in moderately folate deficient rats and by 87% in severely deficient animals (P < 0.05). The combination of folate deficiency and cyclophosphamide reduced the total number of cloning cells further, but ENU alone, or in combination with folate deficiency, did not. These findings indicate that folate deficiency increases the risk of somatic mutations and is lymphocytotoxic in rats. Folate deficiency enhances the mutagenic but not the lymphotoxic effects of ENU, while it increases the lymphotoxic but not the mutagenic activity of cyclophosphamide. Correction of folate deficiency may decrease the immunologic and genetic damage caused by some alkylating agents.

Genetic instability in human T-lymphocytes.

Mutations arising in vivo in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of T-lymphocytes provide a measure of mutation induction in human somatic cells. Studies of measured background HPRT mutant frequency (MF) values show wide inter-individual variation. At the extremes are individuals with 'outlier' MF values, i.e., non-exposed individuals with MF>100x10-6 [Robinson et al., Mutation Res. 313 (1994) 227-247.]. The elevated HPRT MF in one well-studied outlier is due to the in vivo expansion of mutant cells possessing an identical T-cell receptor (TCR) gene rearrangement pattern. We report here that this in vivo expanding TCR clone shows multiple different HPRT mutations and thus possesses a mutator phenotype. Other individuals with T-cell mutator phenotypes have been found, suggesting that this phenomenon may contribute to the extremes of variation in HPRT MFs in the human population.

Molecular bases of hprt mutations in malathion-treated human T-lymphocytes.

Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at the loss of one or more exons. In addition, It was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.

DNA loop domains in a 1.4-Mb region around the human hprt gene mapped by cleavage mediated by nuclear matrix-associated topoisomerase II.

We have mapped the positions in a approximately 1.4-Mb region of genomic DNA around the human hprt gene which are accessible in vivo to cleavage by topoisomerase II associated with the nuclear matrix. These positions, which are interpreted as the boundaries of DNA loop domains, were mapped in K562 cells by examining the truncation of rare-cutter restriction fragments separated by pulsed field gel electrophoresis after topoisomerase II-mediated cleavage, using seven linked markers mapped in this region as probes for indirect end-labeling. Eleven cleavage positions were detected and were interpreted as defining ten loop domains of lengths between 70 and 210 kb (average approximately 135 kb); the hprt gene resides in a 150-kb loop domain. Loop domain boundaries coincided with three of the fifteen deletion breakpoints mapped in a 600-kb sector of this region in human lymphocytes, within the limits of resolution of pulsed field gel electrophoresis; this correlation was not statistically significant.

Radiation quality affects the efficiency of induction and the molecular spectrum of HPRT mutations in human T cells.

Human T lymphocytes can be used to determine the frequency and molecular spectrum of somatic cell gene mutations induced by ionizing radiations both in vivo and in vitro. In vitro exposure of these G0 cells to low-LET 137Cs gamma rays results in the induction of HPRT mutations and a predominant molecular spectrum of DNA deletions and rearrangements, particularly total gene deletions (11-12%). Similar results are found in samples from humans exposed to low-LET radiation from 131I. The doubling dose for mutation induction is calculated to be 0.8 and 1.0 Gy from these exposures performed in vitro and in vivo, respectively. In vitro studies of the effects of high-LET radiation from exposure to 222Rn also showed an induction of HPRT mutations, with a doubling dose of approximately 0.2 Gy. With this radiation, the predominant mutations were small partial deletions, with less than 2% total gene deletions. Studies of humans exposed to high-LET radiation from 239Pu showed an increased HPRT mutant frequency for the group, although no significant dosimetry could be defined. In contrast to the humans exposed to 131I, no increase in the frequency of total gene deletions was found. This is consistent with the results for 222Rn in vitro. The available data show that radiation quality affects both the efficiency of induction and the molecular spectrum of HPRT mutations in human T lymphocytes both in vitro and in vivo. The mutational spectrum may be relatively specific for radiations of different quality and thus allow a more precise measurement of the induction of somatic gene mutations resulting from individual exposures to radiation, and thereby provide more sensitive assessments of health risks.

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents.

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.

Large deletions partially external to the human hprt gene result in chimeric transcripts.

We postulated that gene fusions sometimes occur in normal cells as a result of gene rearrangements as have been observed involving oncogene loci in tumours. To test this, we searched for fusion-gene transcripts in selected human T-lymphocyte large deletion mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using the 3' rapid amplification of cDNA ends (RACE) technique. Aberrant hprt-containing transcripts were observed in seven out of 19 mutants (approximately 36%) indicating that a surprising number of these rearrangements code for processed mRNAs. RNA splicing and polyadenylation occurred downstream of the non-deleted hprt sequence in chimeric transcripts and the majority resulted from mutants with fusions of hprt into regions containing a repetitive element (Alu, LINE or microsatellite).

The aminothiol WR-1065 protects T lymphocytes from ionizing radiation-induced deletions of the HPRT gene.

Aminothiols, such as WR-2721 and its active free thiol, WR-1065, reduce mutations from ionizing radiation in exponentially growing cells. In this study, human noncycling G0 T lymphocytes were exposed in vitro to gamma-irradiation in the presence or absence of WR-1065. The five treatment groups were: (a) control; (b) treatment with 4 mM WR-1065; (c) treatment with 3 Gy of gamma-radiation, from a 137Cs source; and (d) and (e) treatment with WR-1065 30 min prior to or 3 h after 3 Gy of gamma-irradiaiton, respectively. A total of 224 cloned HPRT mutants representing 179 independent mutations were analyzed for genetic alterations using multiplex PCR. Ionizing radiation alone significantly increased the percentage of mutations with gross structural alterations compared to controls (P = 0.02). Although the frequency of such large structural mutations was not different from control cells treated with WR-1065 alone, this aminothiol significantly reduced their frequency among irradiated mutants (P = 0.01) when the radioprotector was present during the irradiation. Addition of WR-1065 3 h postirradiation also greatly reduced the percentage of gross structural alterations; however, due to small numbers, this was not statistically significant. This is the first demonstration that the antimutagenicity of WR-1065 in human cells specifically protects against these kinds of large-scale DNA alterations induced by ionizing radiation. WR-1065 and similar aminothiol compounds may afford protection against radiation-induced mutations through polyamine-like processes, e.g., stabilization of chromatin structure, inhibition of cell proliferation, and influences on DNA repair systems.

Hprt mutations in human T-lymphocytes reflect radioprotective effects of the aminothiol, WR-1065.

Aminothiols such as WR-2721 and its active free thiol WR-1065 have previously been shown to reduce mutations resulting from ionizing radiation in exponentially growing cells. In this study, non-dividing human G0 T-lymphocytes were exposed to the aminothiol radioprotective agent, WR-1065, 30 min before or 3 h after external beam gamma-irradiation and subsequently assessed for survival and mutation induction at the hprt locus. Cytotoxicity due to gamma-irradiation was reduced only when the WR-1065 was present during irradiation. The frequency of hprt mutations, however, was reduced regardless of time of administration, although the reduction was statistically significant only when WR-1065 was added 30 min before irradiation (P < 0.01). This is the first study to demonstrate the protective effects of WR-1065 against radiation-induced mutation in a reporter gene using a human non-cycling cell. Hprt mutations arising in vivo in these cells may be useful for monitoring the radioprotective effect of aminothiols in human populations.

Mitochondrial genome damage associated with cigarette smoking.

We have investigated the level of mitochondrial DNA (mtDNA) damage and deletions in bronchoalveolar lavage tissues from smokers and nonsmokers using quantitative, extra-long PCR and a "common" mtDNA deletion assay. Smokers had 5.6 times the level of mtDNA damage, 2.6 times the damage at a nuclear locus (beta-globin gene cluster), and almost 7 times the level of a 4.9-kb mtDNA deletion compared to nonsmokers, although the latter increase was not significant. Although both genomes (mitochondrial and nuclear) showed significantly increased levels of DNA damage in smokers (mtDNA P = 0.00072; beta-globin P = 0.0056), the relative differences were greatest in the mtDNA. Damage to the mtDNA may inhibit oxidative phosphorylation and, therefore, potentially cause or contribute to chronic lung disease and cancer. Consequently, the mtDNA may be a sensitive biomarker for environmentally induced genetic damage and mutation.