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(Journal of Leukocyte Biology. 2001;69:340-342.)
© 2001 by Society for Leukocyte Biology

DNA damage and apoptosis in mononuclear cells from glucose-6-phosphate dehydrogenase-deficient patients (G6PD Aachen variant) after UV irradiation

Thomas Efferth, Ursula Fabry and Rainhardt Osieka

Department for Internal Medicine IV, University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany

Correspondence: Thomas Efferth, Virtual Campus Rhineland-Palatinate, P.O. Box 4380, 55033 Mainz, Germany.


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ABSTRACT
 
Patients affected with X chromosome-linked, hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency suffer from life-threatening hemolytic crises after intake of certain drugs or foods. G6PD deficiency is associated with low levels of reduced glutathione. We analyzed mononuclear white blood cells (MNC) of three males suffering from the German G6PD Aachen variant, four heterozygote females of this family, one G6PD-deficient male from another family coming from Iran, and six healthy male volunteers with respect to their DNA damage in two different genes (G6PD and T-cell receptor-{delta}) and their propensity to enter apoptosis after UV illumination (0.08–5.28 J/cm2). As determined by PCR stop assays, there was more UV-induced DNA damage in MNC of G6PD-deficient male patients than in those of healthy subjects. MNC of G6PD-deficient patients showed a higher rate of apoptosis after UV irradiation than MNC of healthy donors. MNC of heterozygote females showed intermediate rates of DNA damage and apoptosis. It is concluded that increased DNA damage may be a result of deficient detoxification of reactive oxygen species by glutathione and may ultimately account for the higher rate of apoptosis in G6PD-deficient MNC.

Key Words: flow cytometry • glutathione • polymerase chain reaction • NADPH


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INTRODUCTION
 
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a very common, inborn error of metabolism throughout the world. G6PD converts glucose-6-phosphate to 6-phosphogluconate and reduces nicotinamide adenine dinucleotide phosphate (NADP)+ to reduced NADP (NADPH), which is required by glutathione reductase. Glutathione protects hemoglobin against otherwise detrimental oxidative damage and inhibits apoptosis [1 ]. Because of decreased levels of reduced glutathione, G6PD-deficient patients may experience severe hemolytic crises after infections or after consumption of certain drugs or foods. In contrast to hemoglobin damage in erythrocytes, damage to nucleic acids in nucleated cells from G6PD-deficient subjects is still poorly understood. Depleted G6PD enzyme activities in leukocytes of affected patients have long been recognized [2 , 3 ]. Surprisingly, data on glutathione in nucleated blood cells of G6PD-deficient patients are still not available. As a consequence of a reduced capacity for detoxification by glutathione, DNA of G6PD-deficient patients may, thus, be more vulnerable to DNA damage.

If unrepaired, DNA lesions caused by UV illumination can activate apoptotic pathways [4 ] or neoplastic transformation [5 ]. Although UV light inhibits G6PD activity in Saccharomyces cerevisiae [6 ], its role in G6PD-deficient human beings has not been investigated. Therefore, we studied the effects of UV irradiation on mononuclear cells (MNC) from male G6PD-deficient patients of a German variant, G6PD Aachen, compared with heterozygote females and healthy male donors. DNA strand-breaks in the G6PD gene as well as in another gene not related to the disease, T-cell receptor-{delta} (TCR-{delta}), were measured by means of a polymerase chain reacion (PCR) stop assay. We also measured induction of apoptosis by means of flow cytometry.


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MATERIALS AND METHODS
 
Patients
Three affected males suffering from the G6PD Aachen variant of the enzyme deficiency [7 ] and a G6PD-deficient male from another family that came from Iran were analyzed. Clinical data and laboratory parameters are listed in Table 1 . Four heterozygote females from the G6PD Aachen family and six unrelated healthy male donors served as controls. The G6PD Aachen males were 21–69 years of age, and the heterozygote females, 14–66. The G6PD-deficient Iranian male subject was 13 years old, and the healthy control males ranged in age from 26 to 61 years.


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Table 1. Clinical Data and Laboratory Parameters From G6PD-deficient Patients

The G6PD Aachen variant mutation has been characterized recently at the molecular level [8 ]. The same three patients described in the present investigation were found to be hemizygous at the cDNA position 1089 for a C-G mutation with a predicted amino acid substitution of Asn363Lys. However, this is a unique DNA mutation, which produces the identical amino acid change as the C-A mutation at position 1089 in a Mexican variant, G6PD Linda Loma.

Measurement of cellular glutathione content
Reduced glutathione was measured using a microtiter plate assay according to the manufacturer’s instructions (Cayman Chemical Co., Ann Arbor, MI) [9 ].

UV irradiation
Separated mononuclear cells (1x106 cells/ml) were placed into 35 mm cell culture dishes and illuminated at 312 nm (Benda, Wiesloch, FRG) with 0.08–5.28 J/cm2.

Detection of DNA damage
Because UV-induced lesions in the template DNA decrease the processivity of the DNA Taq polymerase, inhibition of PCR amplification is used to measure DNA damage. PCR-stop assays were performed as described [10 ] with primers for a 1155 bp fragment of the G6PD gene [11 ] and for a 934 bp fragment of the TCR-{delta} gene [12 ]. PCR products were quantified by optical-density scanning (BioRad Gel Doc 1000, Munich, FRG) of ethidium-bromide-stained agarose gels.

Detection of apoptosis
Reduced propidium iodide staining in apoptotic cells is a result of DNA fragmentation and subsequent diffusion of DNA fragments out of the cells. Cell suspensions were stained according to Telford et al. [13 ] and subjected to flow cytometry (Epics-Profile II, Coulter Electronics, Krefeld, FRG). Results were confirmed morphologically by fluorescence microscopy.


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RESULTS
 
The levels of reduced glutathione were significantly lower in G6PD-deficient MNC than in healthy controls (p=0.04; Fig. 1 a ). DNA damage after UV irradiation was monitored in the G6PD (Fig. 1b) and TCR-{delta} genes (Fig. 1c) . UV light induced a dose-dependent cessation of PCR amplification efficacy. There was more DNA damage in MNC from three males of the G6PD Aachen variant and from one G6PD-deficient Iranian male patient than in MNC from six healthy male control subjects (Fig. 1b and 1c) . Intermediate DNA damage was present in MNC from heterozygote females of the G6PD Aachen family (Fig. 1b) . After 24 h post-incubation at 37°C in vitro, apoptosis induced by UV irradiation (0.08–5.28 J/cm2) was more pronounced in MNC from G6PD-deficient males than MNC from heterozygote females or healthy males treated the same way (Fig. 1d) .



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Figure 1. (a) The levels of reduced glutathione in MNC from males of the G6PD Aachen family (#1–#3) were significantly lower than those of healthy control males (#4–#6; p=0.04; mean of three determinations). (b) UV-induced DNA damage in the G6PD gene from three males of the G6PD Aachen family ({blacktriangleup}), from one G6PD-deficient Iranian patient ({blacksquare}), from four heterozygote females of the G6PD Aachen family ({triangleup}), and from six healthy male donors ({circ}). PCR products were standardized as DNA damage per kilobase (mean±SE of three determinations). (c) UV-induced DNA damage in the TCR-{delta} gene (mean±SE of three determinations). For key to symbols, see b. (d) UV-induced apoptosis. The UV-induced rates of apoptosis have been standardized by subtracting the % apoptotic cells in untreated control samples of each patient or volunteer (mean±SE of three determinations). For key to symbols, see b.


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DISCUSSION
 
The rationale for the investigation of nucleated cells derived from long-known observations showing that G6PD deficiency is not restricted to erythrocytes. Apart from hemolytic anemia as the most prominent symptom, several nonhematological manifestations were associated with G6PD deficiency [14 ], although a causative role has not been established.

Although Kahn et al. [7 ] described moderately decreased G6PD levels (76% of normal) in G6PD Aachen leukocytes, we found levels of reduced glutathione in a range from 50% to 70% of normal. We did not explore in-depth the relation between enzyme activity and levels of glutathione. Kahn et al. [7 ] isolated leukocytes from whole blood by gelatin medium and obtained a mixed population consisting of mononuclear and polynuclear cells. We have used Ficoll gradient-centrifugation yielding only MNC. Polynuclear cells, however, contain more glutathione than MNC [15 ]. Decreased amounts of reduced glutathione in MNC indicate that G6PD Aachen is a suitable model to analyze the effect of glutathione for DNA damage and apoptosis in a hereditary disease.

We observed an increased vulnerability to UV-induced DNA damage and apoptosis in MNC from both males of the G6PD Aachen variant and from another G6PD-deficient male originating from Iran. Considering the X chromosome-linked inheritance of G6PD deficiency, healthy male volunteers and heterozygote females of the G6PD Aachen family served as convenient controls. Minimal-to-intermediate-increased rates of DNA damage and apoptosis were found in MNC from these females compared with healthy males. Increased DNA damage and apoptosis in MNC of the G6PD-deficient Iranian male indicate that the observed findings may not be restricted to the rare G6PD Aachen variant. Although we do not regard our data as preliminary, the general relevance of the phenomena described should be investigated in larger populations of G6PD-deficient persons in future studies.

Reduced glutathione is a radical scavenger and prevents DNA damage and apoptosis [1 ]. Low levels of reduced glutathione in G6PD-deficient MNC may not only explain increased DNA damage but also increased apoptosis. The data for UV irradiation presented here are in accordance with the recently detected vulnerability of G6PD-deficient MNC toward DNA-damaging agents such as daunorubicin and ionizing radiation [16 ]. Consistent with these results, G6PD knock-out mouse cells are highly sensitive to oxidative stress [17 ]. Cells transfected with the human G6PD gene displayed higher levels of reduced glutathione and were resistant toward oxidant-mediated cell killing [18 ]. The increased vulnerability of G6PD-deficient cells accords with an investigation of an individual suffering from glutathione deficiency [19 ]. Fibroblasts of this patient displayed increased sensitivity to cisplatinum. Because both DNA damage and apoptosis are involved in pathogenesis, G6PD deficiency may increase the risk of oncogenesis. This has been a matter of controversy for many years. Indeed, a recent, large, epidemiological study supports the view that G6PD deficiency may increase the risk of non-Hodgkin lymphomas [20 ]. A role of the glutathione detoxification system for malignant transformation was substantiated further by genetic polymorphisms of glutathione S-transferase (GST) isoforms [21 ].

Received August 14, 2000; revised December 27, 2000; accepted December 28, 2000.


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