Elevated Sister Chromatid Exchange Research Articles

Role for RAD18 in Homologous Recombination in DT40 Cells

RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

The Bloom's Syndrome Helicase Can Promote the Regression of a Model Replication Fork

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.

The alteration of sister chromatid exchange frequencies in Behçet's disease with and without HLA‐B51

The analysis of sister chromatid exchange (SCE) is a cytogenetic technique used to show DNA damage as a result of an exchange of DNA fragments between sister chromatids. It is known that there is an increased SCE frequency in Behçet's disease (BD). To investigate whether human leucocyte antigen (HLA)-B51-positive patients with Behçet's disease exhibit higher SCE frequencies than those without HLA-B51. Lymphocytes from 75 patients (38 women, 37 men) and from 50 controls (28 women, 22 men) were cultured in darkness for 72 h in the presence of bromodeoxyuridine. Metaphase chromosomes were stained with a fluorescence plus Giemsa technique after a standard harvest procedure. For HLA-B51 typing, DNA was extracted from ethylenediaminetetraacetic acid blood samples and HLA-B5 allele genotyping was performed by the polymerase chain reaction (PCR)-sequence specific primer method. Thirty-nine of 75 patients with BD (52%) and 15 of 50 controls (30%) were found HLA-B51-positive. The SCE frequencies in HLA-B51-positive patients were higher than in HLA-B51-negative ones (P < 0.001), whereas no difference was detected in the control group. This study revealed that there was a significant association between elevated SCE frequencies and existence of HLA-B51 patients with BD.

Biochemical Analysis of the DNA Unwinding and Strand Annealing Activities Catalyzed by Human RECQ1*[boxs

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.

Multiple Roles of Vertebrate REV Genes in DNA Repair and Recombination

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1-/- REV3-/- REV7-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polzeta (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.

UBE2V2 (MMS2) is not required for effective immunoglobulin gene conversion or DNA damage tolerance in DT40

The RAD6/RAD18 heterodimer promotes translesion synthesis via the monoubiquitination of the DNA sliding clamp, PCNA. In S. cerevisiae, a second complex, UBC13/MMS2/RAD5, can extend this single ubiquitin with a non-canonical lysine 63-linked chain. This polyubiquitination step is required for an error-free mode of bypass, possibly template switching by the stalled replication complex. Evidence of a role for the human homologue of MMS2, UBE2V2, in such a process has been inferred from the abrogation of ultraviolet light-induced gene conversion following antisense knockdown of the transcript in human fibroblasts. To ask whether a similar mechanism contributes to abasic site-induced immunoglobulin gene conversion, and to ascertain the role of UBE2V2 in the vertebrate DNA damage response we created a ube2v2 mutant of the chicken cell line DT40. Unlike budding yeast mms2, ube2v2 DT40 does not exhibit significant hypersensitivity to DNA damage, nor the elevated sister chromatid exchange seen in vertebrate rad18 mutants suggesting that UBE2V2 plays a minor or redundant role in RAD18 dependent DNA damage tolerance. In addition, both ube2v2 and rad18 DT40 display more or less normal levels of immunoglobulin gene conversion and, despite the important role played by RAD18 in DNA damage induced translesion synthesis, rad18 DT40, unlike rev1 DT40, does not show a defect in non-templated immunoglobulin gene mutation. Together these data suggest that signalling through PCNA ubiquitination is not required for immunoglobulin diversification in DT40.

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

Genetic Interactions between BLM and DNA Ligase IV in Human Cells

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes: effects on levels of sister chromatid exchanges and chromosomal aberrations

Elevated levels of chromosomal aberrations (CAs) in peripheral blood lymphocytes, widely used as a cytogenetic biomarker of genotoxic effects, have been linked to cancer predisposition. However, tobacco smoking, occupational carcinogen exposure, or time since CA analysis do not appear to explain the cancer predictivity of CAs. Alternatively, the observed CA-cancer association could reflect unidentified exposures or individual susceptibility. We assessed the effects of genetic polymorphisms of DNA repair proteins and xenobiotic-metabolizing enzymes (XMEs) on the levels of CAs and sister chromatid exchanges (SCEs) in peripheral lymphocytes of 145 (CAs) and 60 (SCEs) healthy Caucasians. Genotypes of DNA repair genes X-ray repair cross-complementation group 1 ( XRCC1 codons 194, 280, 399) and 3 ( XRCC3 codon 124), and XME genes glutathione- S-transferase ( GST) M1 and T1 and N-acetyl transferase 2 ( NAT2) were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)-based methods. After Poisson regression adjustment for age, sex, smoking, country, and genotypes, a higher frequency of chromosome-type breaks was observed for NAT2 slow acetylators (in nonsmokers) and GSTT1 null subjects (in smokers). Individuals carrying variant alleles for XRCC1 codons 280 and 194 showed a decreased level of chromosome-type breaks. The effect of GSTM1 null and XRCC1 codon 399 genotypes on the frequency of CAs was modified by smoking. In linear regression models adjusting for age, sex, smoking, and genotypes, none of the polymorphisms significantly affected SCE frequency, although GSTT1 null subjects had a slightly elevated SCE level. Our results are in line with earlier findings on the influence of NAT2, GSTT1, and GSTM1 polymorphisms on the level of lymphocyte chromosome damage and suggest that also XRCC1 polymorphism affects CA frequencies, thus apparently influencing DNA repair phenotype. It remains to be examined whether these or other genetic polymorphisms could explain the observed cancer risk predictivity of high CA frequency.

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

Background: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures. Methods: 40 children ASA classification I–III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3 mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuos propofol infusion at 12 mg/(kg h). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies. Results: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance. Conclusions: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.

Application of the biotin–dUTP chromosome labelling technique to study the role of 5-bromo-2′-deoxyuridine in the formation of UV-induced sister chromatid exchanges in CHO cells

The role of 5-bromo-2′-deoxyuridine (BrdU) in the formation of sister chromatid exchanges (SCEs) in cells exposed to UV radiation was studied. Cells were unifilarily labelled (labelling of one strand of chromosomal DNA) with BrdU or biotin-16-2′-deoxyuridine (biotin-dU) and irradiated in G 1 phase of the cell cycle either with 254 nm, which is absorbed by all nucleobases including bromouracil (BrU) or with 313 nm radiation, which is predominantly absorbed by the BrU moiety. Elevated SCE frequencies were observed in cells irradiated at 254 nm (1.2 and 3.0 J m −2) which were pre-labelled with BrdU or biotin-dU. Following irradiation at 313 nm (38 and 96 J m −2) a statistically elevated SCE frequency was observed in cells pre-labelled with BrdU but not with biotin-dU. In cells pre-labelled with BrdU, UV-radiation at 254 nm was 50–80 times more effective in inducing SCEs than that at 313 nm. This result can be accounted for by the fact that in BrdU-DNA the cross-section for uracilyl radical and bromine atom formation is ∼100-fold higher at 254 nm than that at 313 nm. Upon irradiation at 254 nm, BrdU had a strong sensitising effect on SCE induction: the SCE frequencies observed in cells pre-labelled with BrdU are ∼6 times higher than in cells pre-labelled with biotin-dU. From this it follows that BrdU-induced damage is responsible for more than 80% of the SCEs formed in UV irradiated cells unifilarily labelled with BrdU. Based on photochemical considerations and the fact that chemical agents which form DNA interstrand cross-links are among the most potent inducers of SCEs, we propose that an interstrand cross-link may be the major lesion leading to SCEs in BrdU-labelled cells.

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells.

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.

Sister-chromatid exchange (SCE) frequencies in patients with rheumatoid arthritis: Correlation between SCE frequency and disease activity

AbstractOur objective was to investigate whether sister chromatid exchange (SCE) frequencies in patients with rheumatoid arthritis (RA) are increased and correlate with disease activity. SCE analysis has been performed on metaphase chromosomes obtained from peripheral blood lymphocyte culture in 20 active RA patients and 20 healthy controls. SCE frequencies were higher in active patients than in patients in clinical remission (p < 0.001). Also, SCE values in inactive patients were increased compared with normal controls (p < 0.001). A significant positive correlation was found between SCE frequencies and disease duration, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. In conclusion, the elevated SCE frequencies may be interpreted as an indicator providing genetic predisposition to RA. DNA damage and DNA repair mechanism defects may contribute to the RA process. In addition, SCE analysis may be a useful marker of disease activity and response to the therapy in RA.

The Bloom's Syndrome Protein (BLM) Interacts with MLH1 but Is Not Required for DNA Mismatch Repair

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.

Chromosomes and Stress

The present investigation examines genotoxic effects of prolonged periods of stress; the role of the endocrine system; and the relationship between psychogenic stress and chemical mutagens. Increased levels of both Sister Chromatid Exchanges (SCEs) and Chromosome Aberrations (CAs) were observed in male rats subjected to white noise of either 72 or 240 hrs duration. demonstrating that damage occurs during chronic stress. Rats subjected to foot-shock after having been either hypophysectomized or sham-operated, showed elevation of both SCEs and CAs, indicating that hormones in the hypothalamic-pituitary-adrenal axis do not play a role in genotoxic damage. Rats subjected to foot-shock, and/or the mutagen Mitomycin-C(MMC) showed elevated SCEs, both separately and together. The combined effect was the sum of the individual effects, demonstrating that synergism was not involved. This investigation establishes that psychogenic stress plays a role in genotoxic damage.

Elevated DNA single-strand breakage frequencies in lymphocytes of welders exposed to chromium and nickel.

DNA damage (alkaline filter elution) and sister chromatid exchange (SCE) frequencies were measured in lymphocytes of 39 welders and 39 controls. The welders showed a significantly higher rate of DNA single-strand breakages and significantly elevated SCE values. These results are not in accordance with those of a former study in which only DNA-protein cross-links were measured. The different results may be explained on the basis of different exposure levels for chromium(VI) and nickel. Both methods are not specific but sensitive enough to measure genotoxic damage after occupational exposure to chromium(VI) and nickel in the range of threshold values for the workplace on a collective basis. Additionally, the results indicate that DNA single-strand breakage and DNA-protein cross-links show different increases depending on the exposure levels for chromium and nickel.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Effects of ICRF-193, a catalytic inhibitor of DNA topoisomerase II, on sister chromatid exchange.

To investigate whether mammalian DNA topoisomerase II is directly involved in recombination events, the effects of ICRF-193, a specific catalytic inhibitor on sister chromatid exchange (SCE), were examined in MR-6 cells. ICRF-193 only slightly elevated SCE formation after 3 or 44 h treatments, while VP-16, a cleavable complex forming type of topoisomerase II inhibitor, caused significant enhancement. ICRF-193 had no effect on N-methyl-N'-nitro-N-nitrosoguanidine-induced SCE formation. It would thus appear that the inhibition of topoisomerase II does not affect recombinational repair, and that topoisomerase II inhibitors such as VP-16 and 4'-(9-acridinyl amino) methane sulfon-m-anisidide induce SCE through production of DNA strand breaks, rather than by inhibiting the enzyme activity.

Sister chromatid exchange analysis in workers exposed to noise and vibration

Workers chronically exposed to whole-body vibration and noise are known to develop pathophysiological and psychological disturbances. The frequencies of sister chromatid exchanges (SCEs) and of cells with high frequencies of SCEs (HFCs) were analyzed in lymphocytes of 50 workers occupationally exposed to vibration and noise and of 34 controls. The exposed group included: individuals operating hand-vibrating tools (group 1), ‘test-cell operators’ (group 2) and ‘run-up’ operators (group 3) from an air base and helicopter pilots (group 4). The statistical analysis of the mean SCE count per cell was carried out by multiple regression analysis, comparing various predictor variables: exposure group, duration of exposure, age and cigarette consumption. Only cigarette consumption and exposure group were found to be significantly correlated with the mean SCE frequency. After allowing for the effects of smoking, the analysis indicates that: (1) there was no significant difference between group 1 and controls ( p > 0.05); (2) the differences between group 2 and group 0, group 3 and group 0 and group 4 and group 0 were all highly significant ( p < 0.001); (3) there was no significant difference between groups 2 and 3 ( p > 0.05), nor between groups 2 and 3 combined and group 4 ( p > 0.05); (4) exposure groups 2, 3 and 4 combined, had a significantly elevated mean SCE frequency compared to the control group ( p < 0.0001). Statistical analysis of the proportion of HFCs was consistent with these results. Our data suggest that chronic exposure to whole-body vibration and noise may lead to an increase in the level of SCEs in man. The observed effects may not reflect a direct action of these physical agents on DNA. Alternative explanations may include some of the whole-body vibration and noise-induced or stress-induced pathophysiological alterations which may indirectly induce SCE formation.

Comparison of sister chromatid exchange frequency in peripheral lymphocytes in lung cancer cases and controls

Cytogenetic alterations have been associated with the occurrence of many cancers. However, limited data exist to address whether increased chromosomal changes in surrogate normal tissue are similarly associated with malignancy. As part of an ongoing case-control study of lung cancer, we have studied the factors that affect sister chromatid exchange (SCE) frequency in lymphocytes from lung cancer patients. Further, we sought to investigate whether the factors that affect SCE frequencies were comparable in lung cancer cancer cases and controls. Cases had newly diagnosed, operable primary lung cancer. Controls were friends and spouses of cases. Detailed information on smoking, family history of cancer, medical history, and environmental and occupational exposures was obtained in an interviewer-administered questionnaire. Intake of antioxidants was also determined through the administration of a validated semiquantitative food frequency questionnaire. Metabolic traits studied included the polymorphic glutathione-S-transferase class mu (GST-mu) and variants of P450 isoenzymes CYP1A1 and CYP 2D6. Overall, 78 cases and 78 controls were included in the analysis. Although there was a small number of lung cancer patients who had never smoked in the study (9% of cases), these patients had higher SCE frequencies than current or former smokers. This suggests that factors associated with genomic instability may also play a role in the pathogenesis of lunc cancer. The best fit model for SCE frequency, which had been previously generated from control data alone, included age, gender, smoking, GST-mu, and vitamin A intake. However, when this model was applied to lung cancer patients, smoking was not associated with an elevated SCE frequency. Thus, it is not clear that SCE frequency data in prevalent lung cancer cases and controls are comparable.