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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) 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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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- (TCR-), 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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View this table: [in a new window] |
Table 1. Clinical Data and Laboratory Parameters From G6PD-deficient Patients
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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- 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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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)
.
 View larger version (39K): [in a new window] |
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 ( ), from one G6PD-deficient Iranian
patient ( ), from four heterozygote females of the G6PD Aachen family
( ), and from six healthy male donors ( ). PCR products were
standardized as DNA damage per kilobase (mean±SE of three
determinations). (c) UV-induced DNA damage in the TCR- 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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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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