The association between cardiovascular disease and cancer is well established. Patients with cancer have approximately four times elevated risk for both thrombosis [ [1] Heit J.A. Silverstein M.D. Mohr D.N. Petterson T.M. O'Fallon W.M. Melton 3rd., L.J. Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study. Arch. Intern. Med. 2000; 160: 809-815 Crossref PubMed Scopus (1833) Google Scholar ] and for death given an acute thrombotic event [ [2] Carson J.L. Kelley M.A. Duff A. Weg J.G. Fulkerson W.J. Palevsky H.I. Schwartz J.S. Thompson B.T. Popovich Jr., J. Hobbins T.E. et al. The clinical course of pulmonary embolism. N. Engl. J. Med. 1992; 326: 1240-1245 Crossref PubMed Scopus (975) Google Scholar ] compared to individuals without cancer. Treatment for cancer does not alleviate this risk; in fact, it may increase it, as one in eight cases of venous thromboembolism (VTE) are found amongst patients undergoing chemotherapy [ [3] Khorana A.A. Venous thromboembolism and prognosis in cancer. Thromb. Res. 2010; 125: 490-493 Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar ].

Background: The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis. Objectives: To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs. Patients/Methods: We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor-initiated coagulation in whole blood from individuals with hemophilia and healthy subjects. Results: In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 +/- 0.7 mu m min(-1) to 1.7 +/- 0.4 mu m min(-1) (P = 0.003), 2.3 +/- 0.6 mu m min(-1) to 0.5 +/- 0.1 mu m min(-1) (P = 0.025), and 12.1 +/- 0.7 nm min(-1) to 3.1 +/- 0.7 nm min(-1) (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 +/- 1.2 mu m) prior to clot time (CT) than in healthy individuals (2.6 +/- 0.4 mu m, P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6-fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6-fold (P = 0.001). Conclusions: These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The 'discordance' of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.

Folic acid deficiency (FA−) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA− diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100 mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p = 0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA− diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA− wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA− mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA−) had no effect on cytotoxicity, but FA− diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA− diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.

Dietary supplements are used by most patients with cancer. As nutraceuticals can interact with many drugs, this study investigated the effect of herbal remedies and vitamins on the toxicity of representative cancer chemotherapeutic agents. Fisher 344 rats were fed a standard cereal-based diet or the same diet with additional vitamin E in low (50 mg/kg) or high (750 mg/kg) concentrations, or with added St. John's wort (400 mg/kg). The LD50 was determined after the administration of chemotherapy drugs. Neither low or high vitamin E supplements nor St. John's wort significantly changed the LD50 for doxorubicin, docetaxel, or cyclophosphamide. The nadir white blood cell (WBC) count was significantly higher (P = 0.004) after docetaxel in rats supplemented with low-dose vitamin E, but the drop in WBC count from initial to nadir levels (Nfall) was greater in rats fed a diet containing high vitamin E supplementation (P = 0.04). Similarly, the Nfall was greater in the standard and high vitamin E dietary groups than in the low vitamin E group after cyclophosphamide (P = 0.03). No effect of vitamin E or St. John's wort supplementation occurred on doxorubicin pharmacokinetics. Neither vitamin E nor St. John's wort had an important effect on the mitochondrial deoxyribonucleic acid (DNA) damage caused by either doxorubicin or docetaxel. These data suggest that the leucopenia caused by some chemotherapeutic agents can be modified by dietary supplementation with vitamin E, but the effect seems to be dose-dependent. St. John's wort had neither a beneficial nor a detrimental effect on chemotherapy-induced toxicity.

Mutations arising n viuvo in recorder genes ofhuman blood cells provide biomarkers for mlecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations ofthe glycophorin-A (GPA) or hemoglobin (Hb) genes, nmasured in red blood cells, or mutations ofthe hypoxanthine-guanine phosphoribosyltransferase (hp,t) or EHLA genes, measred in T4ymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x10`) < hprt (5 x 10') = GPA (10 x 10') <HLA (30 x 10 . Mutenposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hpit marker system, about 15% ofbackground mutatin aregrossr al alterations ofthehptgene (e.g., ddetion); theremainderarepoint mutations (e.g., base substitutions or frameshifts). Ionizing rdiations result in doserelated increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions oflinked markers. Possible hpit spectra for defining radiationand chemical exposures are being sought. In addition to their responsveness toenvironmental utagens/carcinogens, three additional fmdings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction ofGPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent ofthe multiple hits required in the evolution ofmalignancy. Finally, fetal hpit mutations frequently have characteristic deletions ofhpltexons 2 and 3, which appear to be mediated by the VDJ recombinase that rearranges the T-cell receptor genes during thymic ontogeny. Illegitimate events such as these also appear to occur in human leukemias.

The conversion of fibrinogen to fibrin and cross-linking to form a stable clot are key processes in effective hemostasis. We investigated fibrinopeptide (FP) A and FPB release, factor XIII (FXIII) activation and fibrin mass in tissue factor initiated coagulation in whole blood from individuals with hemophilia (n=8) and healthy subjects (n=35). For hemophilia whole blood, the rates and patterns of fibrin formation are altered when compared to blood from healthy individuals. The rate of FPA release is decreased from 2.3μ M/min in healthy individuals to 1.5μ M/min in hemophilia and more significantly the rate of FPB release is decreased from 1.3μ M/min to 0.19 μ M/min in hemophilia whole blood. The activated form of FXIII in hemophilia whole blood is formed at a rate of 4nM/min versus healthy individuals where the rate is accelerated to 7nM/min. Prior to clot time in hemophilia whole blood, FPB release is reduced. Whereas more FPA is released in hemophilia whole blood prior to clot time than in whole blood from healthy individuals, 67% versus 47%, respectively. FXIII activation is delayed relative to FPA release. Around clot time in hemophilia whole blood, FPB levels are only ~1μ M and FPA levels are 8 fold higher. In normal whole blood at clot time, FPA release is only 2.5 fold higher than FPB release, potentially suggesting that there is an essential correlation of time dependence between FPA, FPB and FXIII activation to form a stable clot. Whereas, this pattern of fibrin formation is altered in the hemophilia state. Clots from hemophilia whole blood are more soluble as evidenced by a 43% decrease in fibrin mass compared to clots from healthy individuals. Overall, data indicate that hemophilia clots are weaker due to decreased lateral association (FPB release) and are mostly composed of fibrin units that are only partially cross-linked. Thus, suggesting that FXIII activation and FPB release play crucial roles in the pathology of hemophilia and the fragile nature of blood clots in hemophiliacs.

Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R 506 , R 306 and R 679 . Cleavage at R 506 precedes cleavage at R 306 . Cleavage at Arg 306 is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor V Leiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor V Leiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor V Leiden . We observed “APC-resistance” in a patient displaying heterozygous factor V Leiden , Waldenstrom’s macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient’s plasma resistance to APC inactivation was not repaired by immunodepletion of his factor V Leiden and replacement by normal plasma factor V. Conversely when the patient’s fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient’s plasma fVa at R 306 was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor V Leiden , but due to some property which inhibited the lipid dependent cleavage at Arg 306 . The patient’s plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient’s plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient’s plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R 306 . These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.

Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long‐distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3–60 days), then decreases and holds fairly steady to 2 years of age. Environ. Mol. Mutagen. 44:313–320, 2004. © 2004 Wiley‐Liss, Inc.

BACKGROUND. Although patients with malignant disease frequently use dietary supplements, the effects of these agents with regard to chemotherapy are unclear. Therefore, the authors investigated the influence of vitamin B12, folate, and nutritional supplements on chemotherapy-induced toxicity.METHODS. Women with breast carcinoma were asked to complete a questionnaire that recorded their use of dietary supplements. Blood samples were obtained for the assessment of serum vitamin B12 and folate levels before and after the first cycle of chemotherapy and for weekly complete blood counts. Toxicity was evaluated by measuring absolute neutrophil counts and the frequency and severity of oral mucositis.RESULTS. Of the 49 women who submitted questionnaires, 35 (71%) took a combined total of 165 supplements. Compared with patients in a previous study (performed in 1990), patients in the current study had dramatically increased serum folate levels. Initial neutrophil count, but not type of chemotherapy, patient age, or serum vitamin B12 level, was predictive of nadir absolute neutropenia and the decrease from initial neutrophil count to nadir (N-fall) After adjusting for initial neutrophil count, N-fall was found to be lower for women who were taking supplements compared with women who were not taking supplements (P = 0.01) and for women who were taking multivitamins (P = 0.01) or vitamin E (P = 0.03). Women with serum folic acid levels < 20 ng/mL had a smaller decrease in neutrophil count after chemotherapy than did women with higher folate levels (P = 0.04). No significant association between oral mucositis and initial neutrophil count, nadir neutrophil count, N-fall, age, vitamin B12 level, or folate level was found.CONCLUSIONS. The decrease in neutrophil count caused by chemotherapy was ameliorated by dietary supplementation with a multivitamin or vitamin E. In contrast, high serum folate levels were associated with the exacerbation of this decrease in neutrophil count. (C) 2004 American Cancer Society.

Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long-distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3–60 days), then decreases and holds fairly steady to 2 years of age. Environ. Mol. Mutagen. 44:313–320, 2004. © 2004 Wiley-Liss, Inc.

: The purpose of this research project is to better understand the interaction of dietary supplements with cancer chemotherapeutic drugs. This information may be useful to decrease the toxicity and increase the effectiveness of chemotherapy. The scope of the research involves in vivo assessments of nutritional supplement- chemotherapeutic drug interactions and in vitro studies of the mechanisms of nutraceutical- chemotherapeutic interactions. Dietary supplementation of rats with St. John's wort increased the LD5O cyclophosphamide, suggesting that this nutraceutical may decrease the toxicity of cyclophosphamide. Dietary supplementation of rats with 2 levels of vitamin E had no effect on the toxicity of cyclophosphamide. Vitamin E supplementation did not modify rodent hepatic mitochondrial DNA changes caused by docetaxel. In women with breast cancer, the drop in neutrophil count after chemotherapy was less in women taking dietary supplements versus no supplements, and in those taking multivitamins or vitamin E. Our studies suggest that dietary supplements may reduce the toxicity associated with some cancer chemotherapeutic agents used to treat breast cancer.

Mitochondrial DNA (mtDNA) is particularly susceptible to mutation by alkylating agents, and mitochondrial damage may contribute to the efficacy and toxicity of these agents. We found that folate supplementation decreased the frequency of the “common deletion” (4.8 kb, bases 8103–12,936) in liver from untreated rats and from animals treated with cyclophosphamide but not 5-fluorouracil (5-FU). The relative abundance of mitochondrial DNA was greater after chemotherapy but there was no effect of diet. Rats fed with a purified diet had fewer mitochondrial deletions than those maintained on a cereal-based diet after chemotherapy. These results indicate that diet can modulate the extent of mitochondrial damage after cancer chemotherapy, and that folic acid supplementation may be protective against mitochondrial DNA deletions.

Rats fed either a cereal-based or purified diet of variable folate content (deficient, replete, or supplemented) inadvertently were infected with sialodacryoadenitis virus, which resulted in an increased frequency of hepatic mitochondrial DNA (mtDNA) deletions that persisted for three weeks after the period of acute signs of disease. The amount of the "common deletion" (4.8 kb, bases 8103-12937) in liver was measured by quantitative co-amplification of the mitochondrial D-loop and the mitochondrial deletion, using a real-time quantitative polymerase chain reaction assay. The relative abundance of mtDNA was determined by co-amplifying mitochondrial D-loop versus the rat beta-actin gene. Virus-infected rats had more mtDNA deletions (P < 0.0001) and higher copy number (P < 0.0001) than did uninfected animals. There was no effect of diet on frequency of deletions. Diet affected mtDNA relative abundance in the infected, but not the uninfected rats. Relative abundance was higher (P = 0.004) in rats of the high folate group than in rats of the low-folate or folate-replete groups, and was significantly higher in rats of the cereal diet group than that in those of the purified diet group. In conclusion, sialodacryoadenitis virus infection in rats was associated with increased frequency of hepatic mtDNA deletions. Thus, sialodacryoadenitis virus infection mitigated biological processes in the liver of rats, and mtDNA damage was modulated by diet.

The effects of diet and folate status on cyclophosphamide or 5-fluorouracil toxicity were studied in Fischer 344 rats maintained on either a cereal-based diet or a purified diet (AIN-93G). The rats fed the purified diet were divided into 3 groups: folate deficient (no dietary folic acid), folate replete (2 mg folic acid/kg diet), and high folate (2 mg folic acid/kg diet plus 50 mg/kg body weight folic acid intraperitoneally daily). The LD50 for cyclophosphamide was significantly higher for the cereal diet than for the purified diets, but there was no difference among the purified diets. Deaths were predicted by dose, diet, white blood cell count, and BUN on Day 4 after treatment. In the saline-treated rats fed the purified diet, hepatic total glutathione levels increased in the following order: folate deficient < folate replete < high folate. There was no significant difference in aldehyde dehydogenase activities or of microsomal P450 levels in livers from rats on the different diets. In the rats treated with 5-fluorouracil, the high folate rats developed more severe anemia, azotemia, and leukopenia than the other groups. Weight, white blood cell count, hematocrit, and BUN were important predictors of death. The kidneys from rats fed the cereal-based diet were histologically normal, but rats ingesting the purified diet had increasing renal pathology that correlated with folate intake. These results indicate that diet has an important influence on the toxicity of cyclophosphamide and 5-fluorouracil and that folate status modulates hepatic glutathione levels, which is a major cellular defense against oxidant and alkylating agent damage.

Background Monitoring patients on oral anticoagulation is essential to prevent hemorrhage and recurrent thrombosis. We studied tissue factor–induced whole-blood coagulation in patients on warfarin therapy with similar international normalized ratios (INRs). Methods and Results Contact pathway–suppressed whole-blood coagulation initiated with tissue factor was studied in 8 male subjects (group W) and in 1 individual multiple times (subject A). Coagulation profiles for group W showed that subjects with similar INRs had widely varying clot times (6.2 to 23 minutes) and thrombin–antithrombin III (TAT) profiles with rates of 25 to 40 nmol · L −1 · min −1 and maximum levels varying from 192 to 349 nmol/L. The normal control group exhibited clot times of 5.7±0.3 minutes and TAT rates of 57±13 nmol · L −1 · min −1 , reaching maximum levels of 742±91 nmol/L. Subject A, who was stably anticoagulated at an INR of 2.1±0.4 for 6 months, had widely ranging profiles with clot times of 9.0 to 22.7 minutes, TAT maximums varying from 141 to 345 nmol/L, and TAT formation rates of 10 to 57 nmol · L −1 · min −1 . INR did not correlate with TAT formation. Platelet activation was decreased by anticoagulants but also displayed variability. Fibrinopeptide A generation showed threshold variability independent of the INR. Factor VIII levels were increased ( P =0.03) in group W (204±34.4%) compared with normal control subjects (149.4±37.4%). A significant correlation was identified between increasing factor VIII levels and years on warfarin therapy ( r =0.78, P =0.01), suggesting a possible factor VIII compensatory mechanism. Conclusions These results suggest that control of anticoagulation in patients to a set INR therapeutic range may be less secure than anticipated. Patients with similar INRs show significant individual variability in their tissue factor coagulation response, suggesting different risks to anticoagulation when confronted with underlying vascular anomalies.

Summary The influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 μM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 μM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 × 108/ml to 0.5 × 108/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient’s blood was accelerated from ~10 min to 5 min when the platelet concentration increased from 0.05 × 108/ml to 0.11 × 108/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 × 108/ml (range 0.5-3.1 × 108/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 × 108/ml to 3.1 × 108/ml (4.0 ± 0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 × 108/ml. Portions of this work were presented at the 40th Annual Meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, Florida (abstracts #140 and #738).

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.

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.