Gender- and age-related distinctions for the in vivo prooxidant state in Fanconi anaemia patients
Giovanni Pagano1,11,
Paolo Degan2,
Marco d'Ischia3,
Frank J. Kelly4,
Federico V. Pallardó5,
Adriana Zatterale6,
S.Sema Anak7,
Ebru E. Aki
k7,
Gerardo Beneduce1,
Rita Calzone6,
Elena De Nicola1,
Christina Dunster4,
Ana Lloret5,
Paola Manini3,
Bruno Nobili8,
Anna Saviano9,
Emilia Vuttariello1 and
Michel Warnau10
1 Italian National Cancer Institute, G. Pascale Foundation, Via M.Semmola 12, I-80131 Naples, Italy, 2 Italian National Cancer Institute, IST, Genoa, Italy, 3 Department of Organic Chemistry and Biochemistry, Federico II Naples University, Naples, Italy, 4 Division of Life Sciences, King's College, London, UK, 5 Department of Physiology, University of Valencia, Valencia, Spain, 6 Department of Genetics, Elena d'Aosta Hospital, ASL Napoli 1, Naples, Italy, 7 Department of Paediatric Haematology, Istanbul University, Istanbul, Turkey, 8 Department of Paediatrics, 2nd Naples University, Naples, Italy, 9 Department of Paediatrics, Cardarelli Hospital, Naples, Italy and 10 International Atomic Energy AgencyMEL, Monaco
11 To whom correspondence should be addressed Email: gbpagano{at}tin.it
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Abstract
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Some selected oxidative stress parameters were measured in 56 Fanconi anaemia (FA) patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and 173 controls. Untransplanted FA patients showed a highly significant increase in leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG) (P = 0.00003) and a borderline increase (P = 0.076) in urinary levels of 8-OHdG versus child controls. These increases were more pronounced in female FA patients (P = 0.00005 for leukocyte 8-OHdG and P = 0.021 for urinary 8-OHdG). Female FA patients also displayed a highly significant excess of spontaneous chromosomal breaks versus male patients (P = 0.00026), in the same female:male ratio (
1.4) as detected for both leukocyte and urine 8-OHdG levels. Plasma methylglyoxal (MGlx) levels were increased in untransplanted FA patients versus child controls (P = 0.032). The increases in leukocyte and urinary 8-OHdG and in MGlx levels were detected in young FA patients (
15 years), whereas patients aged 1629 years failed to display any differences versus controls in the same age group. A significant increase in oxidized:reduced glutathione (GSSG:GSH) ratio was observed (P = 0.046) in the FA patients aged
15 years, whereas those aged 1629 years, both untransplanted and transplanted, displayed a decrease (P = 0.06) in the GSSG:GSH ratio versus the controls of the respective age groups. No significant changes were detected in plasma levels of vitamin C, vitamin E or uric acid. Transplanted FA patients showed lesser alterations in leukocyte 8-OHdG and in GSSG:GSH ratio versus untransplanted patients. The parents of FA patients displayed a significant increase in plasma MGlx levels (P = 0.0014) versus adult controls. The results suggest a gender- and age-related modulation of oxidative stress in FA patients. The observed increase in urinary 8-OHdG in untransplanted FA patients suggests a proficient removal of oxidized DNA bases.
Abbreviations: BPDS, bathophenanthroline disulfonic acid; CHES, 2-(N-cyclohexylamine)ethanesulphonic acid; DEB, diepoxybutane; dG, deoxyguanosine; FA, Fanconi anaemia; f:m ratio, female:male ratio; HD, healthy donors; HSD, honest significant difference; MGlx, methylglyoxal; MMC, mitomycin C; MPA, metaphosphoric acid; 8-OHdG, 8-hydroxy- 2'-deoxyguanosine; PC, pathology controls; PCA, perchloric acid; TG, total glutathione
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Introduction
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Fanconi anaemia (FA) is a cancer-prone genetic disease characterized by bone marrow failure and susceptibility to myeloid leukaemia and other malignancies. Major aspects of the clinical syndrome include short stature, a set of malformations, endocrinopathies and abnormal skin pigmentation (14).
Mutations that lead to FA fall into 11 complementation groups (57). The FA cellular phenotype is characterized by excessive sensitivity to clastogens, such as mitomycin C (MMC) and diepoxybutane (DEB), which markedly enhance the spontaneous chromosomal instability in FA cells (24). The vast majority of reports in FA research associate FA cell sensitivity to MMC and DEB, as well as the genetic defect(s) in FA, to a deficiency in DNA repair (1,5).
A set of studies have related FA cellular and clinical phenotype to abnormalities in redox pathways (collectively termed oxidative stress). The available evidence for involvement of oxidative stress in the FA phenotype is summarized in Table I (826; reviewed in 4,27,28) and was confined primarily to biochemical and cytogenetic investigations. However, recent reports have focused on the functions of at least the proteins encoded by two FA genes (FANCC and FANCG) (24,29,30), with possible involvement of other FA gene products (4).
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Table I. The involvement of oxidative stress in Fanconi anaemia relies on in vitro and ex vivo cells and body fluids from FA patients, FA protein functions and interactions and toxicity mechanisms of FA-related xenobiotics
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Another line of evidence has related FA cell sensitivity to MMC, DEB and other xenobiotics all characterized by redox-related toxicity mechanisms, including bioreductive biotransformation, redox coupling, glutathione metabolism and induction of oxidative DNA damage, as shown in Table I (23,3137; reviewed in 38). This body of evidence arises from a number studies conducted on several organisms and cell systems and stands against the opinion that MMC and other FA-related xenobiotics exert their toxicity as DNA crosslinkers, which in turn is linked to the established definition of FA as a DNA repair deficiency disorder (38).
A very recent report (25) on the transcription pattern in FANCC-defective versus corrected cells, using a microarray technique, provided evidence for transcriptional regulation of proteins related to the inflammatory response, such as NF
B, cyclooxygenase 2 and HSP70 (39), yet no involvement of transcripts related to DNA repair (25). Concurrent results were obtained in another recent study (26) that identified 69 proteins as direct interactors with FANCA, FANCC or FANCG. These proteins were associated with transcription regulation, signalling, oxidative metabolism and intracellular transport.
Previous reports suggested an in vivo prooxidant state in FA patients (13,15,17,18,21). Thus, the present study was designed to measure a set of oxidative stress parameters in whole blood, plasma, white blood cells and urine from an unprecedented study group of 56 FA patients without or after haematopoietic stem cell transplantation. A group of 54 FA heterozygotes (parents) and a control population consisting of 173 unrelated donors was also recruited. The parameters being measured in the present report included: (i) DNA oxidative damage [leukocyte and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG)] (15,40); (ii) methylglyoxal (MGlx) plasma levels (41,42); (iii) blood glutathione (43); (iv) the plasma levels of some selected antioxidants (vitamin C, vitamin E and uric acid) (44). Additional end-points were evaluated, i.e. spontaneous chromosomal instability, plasma glucose levels (45,46) and glucose 6-phosphate dehydrogenase (G6PDH) activity in red blood cells (47).
The results provided evidence for an in vivo prooxidant state in untransplanted FA patients, with relevant distinctions related to patients' gender and age and, to a more limited extent, in transplanted FA patients and in FA parents.
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Materials and methods
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Study population
A total of 56 FA patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and a total of 173 unrelated controls (108 adults >15 years old and 65 children 115 years old) were enrolled following informed consent as approved by the Ethical Committees of three institutions (2nd Naples University Medical School, ASL Napoli 1 and Cardarelli Hospital, Naples, Italy), in accordance with the Helsinki Declaration of 1975, as revised in 1983. Recruitment required a 24 h urine collection and a 15 ml drawing of heparinized peripheral blood and was carried out thanks to the collaboration of physicians affiliated to six Italian and five Turkish clinical centres (see Acknowledgements). Blood and urine processing was carried out by the project coordinating staff at the INCI, Naples, or by taking the same laboratory consumables and equipment to the recruitment sites in Italy and Turkey. The composition of the groups recruited in Italy and Turkey is reported in Table II, showing superimposable levels in the two national groups as evaluated by the mean levels of leukocyte 8-OHdG, i.e. the parameter with highest number of analyses in the present study. As shown in Table III, the numbers of samples analysed in FA patient groups ranked as: leukocyte 8-OHdG > glutathione > MGlx > plasma antioxidants > urinary 8-OHdG. Patient ages ranged from 1 to 29 years (10.33 ± 5.96 years). The main features of transplanted FA patients are shown in Table IV; at the time of recruitment all of these patients were, to the best of our knowledge, disease free and no major graft versus host disease (GVHD) nor other transplant-related complications were apparent, except for one patient undergoing leukaemic complications who died 15 months after transplantation. Healthy donors (HD) consisted of 42 healthy adult volunteers (aged >15 years) and 65 paediatric outpatients (aged up to 15 years) submitted to diagnostic follow-up for a number of conditions unrelated to any blood disorder, or infectious or malignant disease. An additional control group of adult donors (>15 years), arbitrarily termed pathology controls (PC), was identified based on the superimposability of their analytical results with the HD. The PC group consisted of 65 parents of patients affected by aplastic anaemia, ataxia telangiectasia, Bloom syndrome and xeroderma pigmentosum, whose analytical data were superimposable with those of HD controls. Each sample from patient, parent or control was tagged with a random number and run in blind fashion.
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Table II. Comparison of Italian versus Turkish recruitment groups evaluated by the respective levels (means ± SD) of leukocyte 8-OHdG (mol 8OHdG x 105/mol dG)
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Table III. Numbers of analyses run for the different end-points being measured in untransplanted and transplanted FA patients
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Cytogenetic analysis
To determine spontaneous and DEB-induced chromosomal instability (DEB test), heparinized blood samples were incubated for 72 h at 37°C in RPMI 1640 medium (Eurobio, Les Ulis, France) supplemented with autologous plasma, glutamine and penicillin/streptomycin and stimulated with phytohaemagglutinin. Each culture was set up in duplicate and to one of them was added freshly diluted diepoxybutane (Aldrich, Milwaukee, WI) at a final concentration of 0.1 µg/ml 24 h after culture initiation. Seven hours before harvesting the cells, 5'-bromodeoxyuridine (Sigma, St Louis, MO) was added to the cultures to obtain RBA chromosome banding. Culture harvesting and slide preparation were performed following standard procedures.
Fifty metaphases were analysed from each culture and scored for numerical and structural chromosome abnormalities, taking into account all kinds of rearrangements and breakages, except for gaps (achromatic areas less than a chromatid in width). The results are given as: (i) percentage of aberrant cells (cells with breakages and/or rearrangements); (ii) mean break number per cell; (iii) mean break number per aberrant cell; (iv) percentage of cells with rearrangements. The numerical criteria in scoring spontaneous and DEB-induced chromosomal instability are reported in Table V.
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Table V. Spontaneous (S-CI) and DEB-induced (DEB-CI) chromosomal instability, expressed as per cent cells with breaks, as minimal diagnostic criterium for FA
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In order to avoid any bias due to inter-laboratory differences in cell culture techniques and/or in evaluating chromosomal instability, diagnostic confirmation was run in the same laboratory (A.Z and R.C.) whenever feasible. Italian patients were included in the Italian Fanconi Anaemia Registry (A.Z.).
The centralized cytogenetic confirmation by DEB test was feasible in 28 of 42 untransplanted FA patients, whereas 14 patients failed to be submitted to confirmatory diagnosis, for a variety of reasons (patient's exitus or discontinuing collaboration). Thus, the analytical results of DEB-confirmed and unconfirmed patients were superimposable (P > 0.65; see Table III) in terms of the three most extensively measured parameters (leukocyte 8-OHdG, GSSG:GSH ratio and MGlx) in the two subgroups, prompting the inclusion in the study group of those 14 FA patients only diagnosed at their home institutions.
Determination of 8-OHdG in DNA from leukocytes
Leukocytes from heparinized blood were obtained by Ficoll gradient within 2 h after drawing blood, suspended in phosphate-buffered saline and frozen at 70°C, then shipped in dry ice to Genoa (P.D.). Thawed leukocytes were suspended in 9 vol. of separation buffer (0.32 M sucrose, 10 mM TrisHCl, pH 7.6, 5 mM MgCl2 and 1% Tween 20), then DNA purification was accomplished as reported previously (15). Following nuclease P1 and alkaline phosphatase hydrolysis, DNA samples were filtered through cellulose acetate Centricon filter units (0.22 mm) and separation of 8-OHdG and normal deoxynucleosides was performed in a LC-18-DB (75 x 4.6 mm; Supelco) column equipped with a LC-18-DB guard column cartridge. The solvent system consisted of an isocratic mixture of 90% 50 mM potassium phosphate, pH 5.5, and 10% methanol at a flow rate of 1 ml/min. UV detection was accomplished at 254 nm and electrochemical analysis was carried out with a Coulochem II detector (ESA Inc., Chelmsford, MA). The levels of 8-OHdG were normalized to the amount of deoxyguanosine (dG) detected by UV absorbance at 254 nm. The amount of DNA was determined by a calibration curve versus known amounts of calf thymus DNA. The levels of 8-OHdG were expressed as mol 8-OHdG x 105/mol dG and the 8-OHdG standard used throughout the study was prepared as reported previously (15,40). The amounts of 8-OHdG were quantified by integration of the area of the peak eluted from the electrochemical detector and peak identity was confirmed by co-elution with the standard compound. Analyses were routinely run in triplicate. The coefficient of variation was <15%.
Determination of urinary 8-OHdG
Urine samples were kept refrigerated during the 24 h collection. Thereafter, urine volume was recorded and 50 ml aliquots were frozen in dry ice or in a freezer at 70°C. Samples were shipped frozen in dry ice to London (F.J.K. and C.D.). 8-OHdG was measured in thawed undiluted urine that had been stored for up to 6 months at 70°C. A competitive enzyme-linked immunosorbant assay (ELISA) kit from Genox Corp. (Baltimore, MD) was used. The manufacturer's instructions for use were followed. This involved adding 50 µl of urine per well which had been previously coated with 8-OHdG. A monoclonal antibody specific for 8-OHdG was incubated in the well for 1 h at 37°C and mixed continuously. Any unbound antibody was then removed by washing. A secondary horseradish peroxidase-conjugated antibody was added and the incubation continued for 1 h. The unbound secondary antibody was then removed by washing and a chromatic solution (3,3',5,5'-tetramethylbenzidine) added which develops colour in proportion to the amount of enzyme-linked antibody bound to the plate. The colour reaction was terminated with acid and the absorbance measured at 450 nm. The concentrations of urinary 8-OHdG were calculated from external 8-OHdG standards supplied by the manufacturer. These concentrations were then standardized for creatinine content. All chemicals for urinary 8-OHdG, creatinine, vitamin C and vitamin E (F.J.K. and C.D.) were supplied by Genox (Baltimore, MD), Merck Ltd (Dorset, UK), Sigma Chemical Co. (Dorset, UK) or Rathburn Chemicals Ltd (Peebleshire, UK) and were of the purest grade possible. The coefficient of variation was <5%.
Determination of urinary creatinine
Creatinine was measured (F.J.K. and C.D.) in diluted urine (1 in 20) using a creatinine assay kit (Sigma-Aldrich Chemical Co., Dorset, UK). This basically involves the reaction of creatinine with alkaline picrate, which develops a colour whose absorbance can be measured at 500 nm. The sample is then acidified to identify the presence of any interfering chromogens and re- measured. The concentration of urinary creatinine was calculated from external creatinine standards supplied by the manufacturer.
Determination of MGlx
Plasma from freshly drawn heparinized blood was deep frozen at 70°C and carried in dry ice to M.d'I. and P.M. (Naples). Plasma levels of MGlx were determined according to Espinosa-Mansilla et al. (48). Plasma aliquots (200 µl) were placed in a 3 ml vial, then 300 µl of a solution of 6-hydroxy-2,4,5-triaminopyrimidine (7.02 x 103 M) was added, followed by 200 µl of a 0.02 M sodium acetate solution, pH 4.0, and deionized water up to 2 ml. The samples were heated at 60°C for 45 min, filtered through a 0.45 µm nylon filter and 100 µl aliquots were injected into an HPLC. HPLC-FD determinations were performed with a Gilson instrument equipped with a model 305 pump. A Jasco FP-110 fluorescence detector with excitation at 352 nm and emission at 447 nm was used for detection.
Analyses were carried out on a 250 x 4.60 mm Sphereclone ODS (reverse phase) 5 µm column using 0.02 M sodium acetate (pH 4.0) and acetonitrile (99:1 v/v) as eluant at a flow rate of 1.3 ml/min. Calibration curves for 6-methylpterin (0.881.9 µM) and the standard addition method allowed calculation of MGlx levels by integration of peak areas. The coefficient of variation was <35%.
Determination of oxidized glutathione and total glutathione
Blood samples (0.5 ml) were quickly treated, immediately after venipuncture, with 12% (v/v) perchloric acid (PCA) containing 2 mM bathophenanthroline disulfonic acid (BPDS) (1:5) to determine total glutathione (TG). In order to determine GSSG levels, blood (0.5 ml) was acidified immediately after being drawn with 0.5 ml of 12% PCA, 2 mM BPDS and 40 mM N-ethylmaleimide (43). Samples were immediately deep frozen at 70°C and shipped in dry ice to Valencia (F.V.P. and A.L.). After thawing, acidified blood was centrifuged at 15 000 g for 5 min at 4°C. The supernatant, free of proteins, was used for the determination of GSSG. Afterwards, 20 µl of 0.2% Metacresol Purple were added and the pH was set at 89 with 3 M KOH and 0.3 M 2-(N-cyclohexylamine)ethanesulphonic acid (CHES). Then the sample was centrifuged at 15 000 g for 5 min at 4°C. A 25 µl aliquot of the supernatant was taken and poured into a crystal tube containing 50 µl of a 1% fluordinitrobenzene solution in ethanol. Finally, the mixture was incubated for 45 min at room temperature and in darkness, then the sample was vacuum dried up to 70 mtorr. Before injection into the chromatograph the precipitate was re-diluted in 200 µl in methanol/water (80/20 v/v).
The protein-free supernatant was used for determination of TG. A 20 µl aliquot of 1 M iodacetic acid in 0.2 mM m-cresol purple solution was added to 0.2 ml of the supernatant and this mixture was brought to a pH of 89 with 3 M KOH + 0.3 M CHES, then incubated for 10 min in the dark at room temperature. Then 400 µl of 1% fluordinitrobenzene in ethanol were added and this solution was incubated for 24 h in the dark at 4°C. The samples were centrifuged at 15 000 g for 10 min at 4°C and then injected into the chromatograph.
Determination of ascorbic acid and uric acid
The HPLC analysis of uric acid and ascorbic acid was based on the method of Iriyama et al. with modifications (49). Plasma was previously acidified 1:1 with ice-cold 10% metaphosphoric acid (MPA), then was centrifuged and the supernatant stored at 70°C ready for shipment on dry ice to King's College London (F.J.K. and C.D.). This supernatant was thawed on ice and diluted 2:3 with ice-cold 5% MPA. To this was added 100 µl of HPLC grade heptane by mixing on a vortex stirrer for 40 s, the samples were centrifuged at 13 000 r.p.m. for 5 min at 4°C and the lower (aqueous) layer was removed and again treated with heptane until the supernatant was clear. This supernatant was transferred to a 0.8 ml HPLC vial. Aliquots of 20 µl were injected onto a 4.6 x 250 mm, 5 µm C18 Apex II column with guard (Jones Chromatography, Glamorgan, UK) and eluted with a 0.2 mol/l K2HPO4H3PO4 (pH 2.1) mobile phase containing 0.25 mmol/l octane sulfonic acid at a flow rate of 1.0 ml/min. An electrochemical detector (EG & G Instruments, Wokingham, UK) was used for detection, with the working electrode set at 810 mV and a sensitivity of 0.2 or 0.5 µamp. Final concentrations for ascorbic acid and uric acid were calculated with external standards which were run simultaneously. The coefficient of variation of analysis was <5%, with a minimum detection limit for ascorbic acid of 0.5 µM and uric acid of 0.1 µM.
Determination of
- and
-tocopherol
Vitamin E (
- and
-tocopherol) was measured in plasma based on the method of Kelly et al. (44) using HPLC with ultraviolet detection (F.J.K. and C.D.). An aliquot of 100 µl of thawed plasma was mixed with 5 µg internal standard (
-tocopherol acetate) in 100 µl of ethanol. Ice-cold HPLC grade hexane (400 µl) was added to the plasma and vortexed twice for 40 s. Following centrifugation at 3000 r.p.m. for 5 min at 4°C, the hexane layer was carefully removed into 0.8 ml HPLC vials and evaporated to dryness under a stream of nitrogen. The extract was then redissolved in 400 µl of HPLC grade methanol and vortexed for 2 x 40 s. Aliquots of 100 µl were injected onto a 4.6 x 100 mm 5 µm C18 column Apex II column with guard (Jones Chromatography) and eluted with 98% HPLC grade methanol at a flow rate of 1 ml/min. Final concentrations for
- and
-tocopherol were calculated with external standards and adjusted for recoveries with the internal vitamin E standard. The coefficient of variation of analysis was <5%, with a minimum detection limit for both tocopherols of 0.15 µM.
All chemicals were supplied by Merck (Dorset, UK), Sigma Chemical Co. (Dorset, UK) or Rathburn Chemicals (Peebleshire, UK) and were of the purest (usually HPLC) grade possible.
Determination of G6PDH and glucose
Measurement of G6PDH activity from frozen and thawed red blood cells was carried out (G.B.) using the assay kit available from Randox (Ulster, UK) (50). This involves the reaction of glucose 6-phosphate with NADP+, which causes an absorbance increase that can be measured at 340 nm. The activity of G6PDH was referred to haemoglobin concentration, which was measured by the methamoglobin method (51).
Glucose plasma levels were measured with a Dimension® assay kit provided by Dade Behring Inc. (Newark, DE), based on an adaptation of the hexokinase/glucose 6-phosphate dehydrogenase method (52).
Statistical analyses
Differences between groups were tested using one-way analysis of variance (ANOVA) followed by the post hoc Tukey honest significant difference (HSD) test (M.W.). When data sets with unequal n were considered, the SpjotvollStoline test (Tukey HSD for unequal n) was used. For comparisons of data expressed as percentages, an arcsin transformation (x' = arcsin[
(x/100)]) was performed prior to testing in order to respect the data normality prerequisite (53). The level of significance was set at
= 0.05. All analyses were performed using statistical routines in Statistica© 5.1 software.
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Results
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Figure 1 shows the data for 8-OHdG determinations in leukocytes from FA patients of all ages, their parents and controls. A very highly significant excess of 8-OHdG (P = 0.00003) was observed in 40 leukocyte samples from untransplanted FA patients versus 36 leukocyte samples from child controls. Considering the age groups of FA patients, as shown in Table VI, excess levels of leukocyte 8-OHdG were observed to a greater extent for 16 female than for 16 male untransplanted FA patients (aged
15 years), with an
2-fold increase in leukocyte 8-OHdG levels (5.77 ± 2.96 mol 8-OHdG x 105/mol dG) versus young (
15 years) female controls (2.28 ± 1.01 mol 8-OHdG x 105/mol dG, P = 0.00003). It was noted, surprisingly, that a group of 8 adolescent and adult FA patients (aged 1629 years) failed to show the abnormalities in 8-OHdG levels observed in younger patients (Table VI). This group of older untransplanted FA patients displayed 8-OHdG levels that coincided with the values observed in 12 controls selected in the same age range (Table VI). The results for leukocyte 8-OHdG in 18 transplanted FA patients of all ages showed only a non-significant increase versus controls (Figure 1). However, when the two age groups (
15 and 1629 years) were examined, as in untransplanted patients a dramatic difference was observed in 13 transplanted patients aged
15 years versus 5 transplanted patients aged 1629 years, in that the former (especially female patients) showed a significant increase in 8-OHdG (4.88 ± 3.76 mol 8-OHdG x 105/mol dG) that averaged close to the data observed for untransplanted female patients (5.77 ± 2.96 mol 8-OHdG x 105/mol dG), versus the control value of 2.28 ± 1.01 mol 8-OHdG x 105/mol dG (Table VI). In contrast, a group of 5 transplanted patients aged 1629 years displayed leukocyte 8-OHdG levels that were superimposable on the controls in the same age range (Table VI). It was interesting to note that the female:male (f:m) ratio in leukocyte 8-OHdG levels in young (
15 years) FA patients (both untransplanted and transplanted) ranged from 1.36 to 1.54, unlike both controls and adolescent/adult FA patients, where the f:m ratio was close to 1 (0.811.15). Leukocyte 8-OHdG in 49 leukocyte samples from FA parents failed to display any excess levels (2.88 ± 1.22 mol 8-OHdG x 105/mol dG), compared with 58 leukocyte samples from adult controls (3.39 ± 1.45 mol 8-OHdG x 105/mol dG), as shown in Table VI, and with the same f:m ratio as in controls, close to 1.

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Fig. 1. Leukocyte 8-OHdG levels in female versus male patients, parents and controls. Data were from 23 plasma samples from untransplanted and 7 samples from transplanted FA patients versus 18 samples from their parents and controls (11 adult and 12 child controls).
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The measurements of urinary 8-OHdG levels showed a non-significant, although borderline (P = 0.076), increase in 8-OHdG levels in the urine samples from 13 untransplanted FA patients versus 47 controls (Figure 2). However, 7 female FA patients (aged
15 years) displayed a significant increase in urinary 8-OHdG levels (7.31 ± 2.36 nmol 8-OHdG/mmol creatinine) versus 10 controls in the same age range (4.46 ± 0.53 nmol 8-OHdG/mmol creatinine) (P = 0.02) or versus 26 female controls of all ages (4.7 ± 0.53 nmol 8-OHdG/mmol creatinine) (P = 0.0014), as shown in Figure 2. The urinary 8-OHdG levels in 2 male FA patients in the same age range (5.48 ± 1.41 nmol 8-OHdG/mmol creatinine) displayed a non-significant increase versus controls. Again, the f:m ratio for FA patients (1.33) was close to that observed for leukocyte 8-OHdG levels. A decrease in the urinary 8-OHdG levels in 4 male untransplanted patients aged >15 years (3.08 x 0.23 nmol 8-OHdG/mmol creatinine) suggested an analogous normalization as seen for leukocyte 8-OHdG over the age of 15 years. The urinary 8-OHdG levels in 20 parents of FA patients were superimposable on control data. No data were available for urinary 8-OHdG in transplanted FA patients.

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Fig. 2. Urinary 8-OHdG levels in 13 untransplanted FA patients (males versus females), 24 of their parents and 47 controls of all ages (child and adult controls showed superimposable 8-OHdG urine levels).
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Concomitant with 8-OHdG, a highly significant gender difference was also observed in spontaneous chromosomal instability, expressed as per cent of cells with chromosomal breaks. As shown in Figure 3, the data from the patients recruited in the present study and records from the Italian Fanconi Anaemia Registry (RIAF) showed that 27 female FA patients exhibited a highly significant excess rate of spontaneous breaks (24.44 ± 14.39) versus 25 male patients (15.56 ± 7.86) (P = 0.00026), as opposed to 169 male and 180 female controls from RIAF records which showed superimposable rates of spontaneous chromosomal breaks (3.72 ± 3.03 and 4.20 ± 3.28, respectively). The data on DEB-induced chromosomal instability failed to show any significant gender difference (data not shown). The f:m ratio for spontaneous chromosomal instability rates in FA patients was 1.57, overlapping the 1.54 f:m ratio observed for leukocyte 8-OHdG levels detected in young untransplanted FA patients (Table VI). An overall comparison of the mean f:m ratios for leukocyte and urinary 8-OHdG levels and spontaneous chromosomal instability in FA patients versus controls, from data shown in Table VI and Figures 2 and 3, showed a highly significant difference (P = 0.0004) in mean f:m ratios of 1.47 ± 0.10 (n = 4) for FA patients and 1.02 ± 0.11 (n = 8) for controls.

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Fig. 3. Spontaneous chromosomal instability (expressed as per cent cells with chromosomal breaks) in FA patients (27 females and 25 males) versus 349 controls from RIAF records (DEB-negative subjects).
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The concentrations of MGlx showed a highly significant difference among the sampling groups (PANOVA = 5 x 106). The plasma levels of MGlx in 23 untransplanted FA patients showed a non-significant increase versus 34 controls, as shown in Figure 4. However, on splitting the data into two age groups (
15 and 1629 years), the increased MGlx levels were higher in the child patient group versus the adolescent/adult group, as shown in Table VII. In particular, 7 female patients (aged
15 years) showed MGlx levels of 543.06 ± 283.65 nM versus 384.47 ± 58.76 nM MGlx for 12 child controls. Again, 5 older patients displayed lower MGlx levels that were superimposable on 11 controls of the same age group (1629 years), or lower than controls on considering and removing the exceedingly high MGlx level of a 20-year-old patient who had received frequent blood transfusions (Table VII). A noteworthy finding was that plasma from 18 FA heterozygotes showed a significant increase in MGlx levels (627.07 ± 142.81 nM) versus 11 adult controls (450.13 ± 103.40 nM) (P = 0.0014), higher than those observed in FA patients (Figure 4). No increase in MGlx levels was detected in 7 transplanted FA patients. The data for glycaemia, related to MGlx formation, showed a significant increase in untransplanted FA patients (P = 0.032) and a non-significant increase in FA heterozygotes versus their respective control groups (Table VIII).

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Fig. 4. MGlx plasma levels in 23 untransplanted, 7 transplanted FA patients and 18 FA parents versus 11 adult controls and 12 child/adolescent controls. No gender difference was detected.
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The levels of TG, GSSG, GSH and the GSSG:GSH x 100 ratio in whole blood from 23 untransplanted FA patients and in a group of 12 transplanted FA patients of all ages failed to show any significant difference versus their parents and controls (data not shown). When the data were divided in the two age groups (
15 and 1629 years) and referenced to the corresponding controls the GSSG:GSH ratio was the same in both untransplanted and transplanted FA patients, depending on age group, as shown in Figure 5. A significant increase in GSSG:GSH x 100 ratio was observed in 28 FA patients aged
15 years (9 transplanted and 19 untransplanted FA patients) (GSSG:GSH x 100 = 3.10 ± 2.69) versus 43 controls in the same age group (GSSG:GSH x 100 ratio = 2.01 ± 1.80) (P = 0.046). In contrast to younger patients, a group of 7 FA patients aged 1629 years (4 untransplanted, 3 transplanted) displayed an unexpected and dramatic decrease in GSSG:GSH x 100 ratio (0.63 ± 0.41) versus 11 controls in the same age range (3.46 ± 3.77). The limited numbers of patients and controls caused this difference to be non-significant, although borderline (P = 0.06) (Figure 5).

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Fig. 5. (A) Young FA patients (19 untransplanted and 9 transplanted patients), up to the age of 15 years, showed higher GSSG:GSH x 100 ratio than 43 controls in the same age range. (B) A dramatic decrease in GSSG:GSH x 100 ratio was observed in 7 FA patients aged 1629 years versus 11 controls in the same age range.
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Measurements of G6PDH activity, known to affect glutathione status, failed to show any differences in FA patients versus their parents or controls (data not shown). Plasma levels of ascorbic acid,
- and
-tocopherol and uric acid in plasma from FA patients and from their parents failed to result in any significant changes compared with controls (data not shown).
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Discussion
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The present study provides evidence for an in vivo prooxidant state in untransplanted FA patients with relevant distinctions related to patients' gender and/or age, also pointing to a gender-based distinction in spontaneous chromosomal instability. Post-transplant patients and FA parents also exhibited some hallmarks of an in vivo prooxidant state, although to a lesser extent than untransplanted FA patients. The numbers of FA patients and parents recruited in this study exceed any population of FA families recruited in any analogous study, to the best of our knowledge. Albeit lacking patients' assignments to complementation groups, this report provides a realistic dataset of the inter-individual variability of oxidative stress parameters in FA patients and heterozygotes. Moreover, the homogeneous procedures for sample processing minimized any underlying bias related to variability in sample handling, whether samples were processed in centralized facilities by the same personnel using the same materials on samples from distant donors or on ad hoc expeditions throughout Italy and Turkey.
The results corroborate previous evidence associating FA cellular and clinical phenotype with oxidative stress (15; reviewed in 4,27,28). Implications for likely endocrine factors are suggested by the data on gender differences (both leukocyte and urinary 8-OHdG, along with spontaneous chromosomal instability) and on age group distinctions between pre- and post-pubertal patients, observed for leukocyte 8-OHdG, MGlx and glutathione levels (see Tables VI and VII and Figure 5).
Whether the observed alterations in glutathione balance may be attributed to a primary deficiency in some FA gene product or to the prooxidant state due to other upstream effectors is an open question, although the two hypotheses are not mutually exclusive. In favour of the former hypothesis, a report by Cumming et al. (30) demonstrated that FANCC protein exerts redox-associated regulation of a glutathione S-transferase (GSTP1). Since FANCC is part of the FA protein complex, it might be envisioned that an abnormality in the association between FANCC and GSTP1 might be related to a deficiency in the protein complex affecting the in vivo glutathione status (4). The present data on glutathione imbalance may be relevant in the clinical management of FA patients, as it could be suggested that glutathione status might be analysed in patients' blood and that ad hoc clinical trials might be undertaken to monitor, and possibly improve, glutathione status in FA patients.
The data on MGlx are the first report in FA of this product of oxidative carbohydrate and amino acid degradation and a marker of diabetes-associated oxidative stress. A significant increase in MGlx levels has been detected in plasma from young (
15 years old) FA patients, consistent with excess glycaemia levels in FA patients (45,46) and confirmed in the present study (Table VIII). The lower MGlx levels in transplanted patients versus untransplanted patients might be related to the association of MGlx with red blood cell functions (54) pertaining to the transplant donor. The significantly elevated MGlx levels in 18 parents of FA patients, if confirmed on more extensive groups of FA heterozygotes, may suggest that MGlx analysis might be developed as a candidate diagnostic test for the FA heterozygous state, which is presently lacking.
The present results may lend support to a mechanistic link between MGlx levels and other features of oxidative stress in FA, e.g. glutathione imbalance and oxidative DNA damage, as well as with some relevant clinical features of FA, e.g. endocrinopathies, including hyperglycaemia up to overt diabetes in FA patients and their families (45,46). These complications or clinical features have been regarded so far as scattered events lacking any mechanistic relationship to the main focus of haematopoietic impairment. The formation and/or toxicity of MGlx has been associated with diabetes (55,56), osteoarthritis (57) and premature atherosclerosis in renal failure patients undergoing dialysis (58). Moreover, MGlx has been shown to be involved in GSH depletion (56) and associated with DNA and protein cross-linking (57). Thus, the present results of MGlx analyses are consistent with the data on glutathione imbalance and 8-OHdG accumulation in FA patients. The recognized toxicity of MGlx towards pancreatic ß-cells may offer a link between the prooxidant state in FA and the propensity of FA patients to develop a diabetes-prone condition (54,55). It is well established that diabetes, as for example cancer, is one of the pathological conditions displaying a direct relationship with oxidative stress (59,60). Thus, the present data for MGlx analyses may relate to several clinical features of FA based on an underlying prooxidant state.
The results of leukocyte 8-OHdG analyses corroborate our previous report (15), by confirming excess 8-OHdG levels in FA patients versus controls. These excesses were observed to a significantly greater extent in female than in male patients and in child patients versus adolescent/adult patients. Interestingly, these distinctions were maintained in both pre- and in post-transplant patients. The role of oxidative DNA damage in carcinogenesis (61), ageing (62) and in a number of other pathologic processes is well established. Thus, excess 8-OHdG levels in FA are consistent with cancer proneness in this disorder. Consistent with leukocyte 8-OHdG accumulation, urinary 8-OHdG levels were also significantly increased in untransplanted young female patients, whereas young male patients showed a slight, though non-significant, increase versus controls. Older patients (1629 years) displayed 8-OHdG levels (both leukocyte and urinary 8-OHdG) overlapping the respective control data. One might expect to find a decrease in urinary 8-OHdG levels, on the assumption that defective DNA repair would result in decreased removal of damaged DNA bases, hence in a lower than normal urinary excretion of 8-OHdG (63). The results failed to support a deficiency in removing oxidatively damaged DNA, thus casting doubts about the in vivo relevance of the established theory associating FA with defective DNA repair (1,5).
Consistent with our previous report (15), a direct association has been found between oxidative DNA damage and chromosomal instability as hallmarks of FA phenotype. It was intriguing to note that the f:m ratio for spontaneous chromosomal instability was the same as observed for leukocyte 8-OHdG levels in young (
15 years) untransplanted FA patients, and close ratios were also observed in leukocyte 8-OHdG from young transplanted FA patients and in urinary 8-OHdG from untransplanted FA patients, whereas no such gender distinction was detected in 8-OHdG levels from adolescent/adult FA patients nor in parents nor in controls.
It may be suggested that the gender- and age-associated differences observed in oxidative stress parameters in FA may be related to the mechanisms of action of androgens and estrogens, which, in turn, are known to be related to redox-dependent biotransformation mechanisms, making these hormones recognized modulators of oxidative stress (6466). Androgens affect glutathione levels (64) and puberty has been shown to be involved in the regulation of phospholipid hydroperoxide glutathione peroxidase (67). An involvement of sex hormones in FA pathogenesis may suggest a mechanistic explanation for the benefits observed in FA patients undergoing androgen administration.
A surprising outcome of the present study was the failure to detect any abnormalities in the concentrations of plasma antioxidants, i.e. vitamin C, vitamin E and uric acid, in FA patients. No explanation can be provided to date for this finding, which awaits adequate interpretation.
The present study points to prooxidant state as a key event in the clinical phenotype of FA, providing mechanistic links between oxidative stress and disease progression. The observed gender- and age-related distinctions in oxidative stress parameters both prevent generalizations about the occurrence of a prooxidant state in FA and disclose avenues for further laboratory and clinical investigations on FA. A unifying interpretation is here proposed for the cellular and clinical features of FA, encompassing chromosomal instability, bone marrow failure, malignancies and a set of as yet poorly explained side events, such as malformations, endocrinopathies and anomalies in skin pigmentation. This might lead to a reappraisal of this disorder as a dysmetabolic disease, as far as FA patients, especially young female patients, are concerned, with an in vivo prooxidant state that affects a number of physio-pathological phenomena not confined to haematopoietic impairment. This reappraisal might lead to innovations in FA diagnosis, as well as in the management of FA patients, in the prospect of appropriate clinical trials focused on the chemoprevention of disease progression.
 |
Acknowledgments
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This study was made feasible by the Contributors to the EUROS (European Research on Oxidative Stress) Project, Objective 1, who were as follows. Clinicians contributing to patient and control recruitment: Stefano Calvieri, Duran Canattan, Kaan Kavakl
, Yurdanur K
l
n
, Daniela Longoni, Maria A.Pisanti, Walter Pörnbacher, Nevin Yalman, Akif Ye
ilipek, Marisa Zaccagnini and Alfonso Zaccaria. Sample processing and analyses: Margherita Cerrone, Paolo Ciavolino, Germana De Stefano, Francesca Gallucci, Giacomo Iazzetta and Claudio Polese. Project organization and data processing: Antonio Marfella, Francesca Panzeri and Virginia Rossi. The present study, a part of the EUROS Project, was supported by the European Commission, DG XII, contract no. BMH4-CT98-3107, and by the Italian Association for Fanconi Anaemia Research (AIRFA).
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Received December 24, 2003;
revised May 14, 2004;
accepted May 16, 2004.