Review Article
Detection of Clastogenic Factors in Oxidative Stress-Associated Diseases.
Usefulness of this Assay for the Evaluation of Anti-oxidants(1)
Ingrid Emerit
Institut de Santé et Développement Université Pierre
& Marie Curie
15, rue de l’Ecole de Médecine 75006 Paris, France
Tel./Fax: 33-(0)143299939
(1) This work has been presented at the “International Free Radical
Research Conference”
Gödöllõ, Hungary, 25–27 August 1997.
CEJOEM 1998, Vol.4. No.1.:3-10
Abstract
Clastogenic i.e. chromosome damaging substances are present in the plasma
of patients with a variety of pathological conditions accompanied by oxidative
stress. These include irradiation exposure, chronic inflammatory diseases
of the connective tissue, gut, liver, nervous system, skin etc., HIV-infection,
ischemia reperfusion injury, as well as the congenital breakage syndromes.
The formation of these so called clastogenic factors
(CF) and their damaging effects are mediated by superoxide, since superoxide
dismutase (SOD) is regularly protective. The strongest evidence for the
role of O2 came from in vitro experiments, in which cells were exposed
to superoxide-generating systems. The supernatant of these cells contained
CF, while cell-free systems did not result in CF formation. It was also
shown that CF stimulate the production of superoxide by monocytes and neutrophils.
The fact that CF are produced via superoxide and stimulate themselves superoxide
leads to a vicious circle and to a chronic pro-oxidant state. Biochemical
analysis has identified lipid peroxidation products, arachidonic acid metabolites,
nucleotides of inosine and cytokines, in particular tumor necrosis factor,
as the clastogenic components of CF. Correlations were observed between
CF and other biomarkers of oxidative stress such as increased spontaneous
superoxide production by neutrophils and decreases in plasma thiols. Due
to their chromosome damaging effects, these oxidants can be detected by
classical cytogenetic techniques. The CF assay is useful for the evaluation
of the efficacy of anti-oxidant treatments. The results of two pilote studies
conducted with plant anti-oxidants in persons irradiated as a consequence
of the Chernobyl accident showed that the clastogenic activity in the plasma
of the treated persons is reduced to normal levels and that the benefit
of the treatment persists up to one year after arrest of the treatment.
Key words:
Oxidative stress, clastogenic factors, CF assay, free radical-related diseases,
antioxidant treatment
Abbreviations:
CF = Clastogenic factor(s)
HIV = Human immune deficiency virus
SOD = Superoxide dismutase
TNF = Tumor necrosis factor
ACS = Adjusted clastogenic score
Received: 12 November 1997
Accepted: 16 December 1997
Introduction
Clastogenic i.e. chromosome damaging substances are present in the plasma
of patients with a variety of pathological conditions accompanied by oxidative
stress. These include irradiated persons, patients with chronic inflammatory
diseases of the connective tissue, the gut, the liver or the nervous system,
HIV-infected patients, as well as the hereditary chromosomal instability
syndromes (Bloom’s syndrome, Fanconi anemia and ataxia telangiectasia (for
review see Emerit 1994). Also after ischemia-reperfusion injury, the plasma
of patients contains clastogenic activity (Emerit et al., 1995a). The formation
of these breakage factors or clastogenic factors (CF), as well as their
chromosome damaging effects, are mediated by the superoxide anion radical,
since they are regularly inhibited by superoxide dismutase (SOD). For this
reason, the term “superoxide-mediated clastogenesis” was proposed (Emerit
et al., 1996). Our experiments with fluorescently labelled SOD indicated
that the enzyme protects the cells not only by dismutating extracellular
superoxide, but that it also binds to the cell surface, in particular to
monocytes/macrophages. The superoxide production of these cells is diminished,
what results secondarily in diminished CF formation. Superoxide is not
a direct DNA-damaging agent, but an initiator of a series of events leading
to the formation of clastogenic materials. Biochemical analysis of CF preparations
identified three major classes of chemical substances: i) lipid peroxidation
products such as hydroperoxides, malondialdehyde and 4-hydroxynonenal,
derived from arachidonic acid of membranes (Emerit et al. 1991), ii) cytokines,
such as tumor necrosis factor alpha (Emerit, 1994) and iii) unusual nucleotides
such as inosine di- and triphosphate (Auclair et al., 1990; Emerit et al.,
1997a). The clastogenic properties of these components were confirmed with
the respective commercial standards. The superoxide-stimulating properties
of CF preparations from various sources could be confirmed with the cytochrome
C assay (Emerit, 1990a) and with chemiluminescence studies (Emerit et al.,
1995a). TNF alpha and inosine triphosphate may be responsible herefor.
CF formation via superoxide generation and superoxide generation via performed
CF create a vicious circle responsible for persistent oxidative stress
and longterm genotoxic effects.
The CF assay
The methods used for detection of these endogenous clastogens are the same
as those currently used for various exogenous clastogenic agents. Since
it is known that the clastogenic activity is in the small molecular weight
fraction, the plasma is ultrafiltrated through a Millipore or Amicon ultrafiltration
filter. In the initial description of the technique (Emerit, 1990b), filters
with a cut off at 10,000 daltons were recommended. Afterwards, when TNF
was recognized as one of the clastogenic components, they were replaced
by filters with a cut off at 30,000 daltons. The ultrafiltration step is
useful for elimination of all high molecular weight materials, which might
disturb culture growth due to blood group incompatibilities.
For evaluation of the clastogenic effects of a plasma
sample, regular blood cultures are set up with whole blood from a healthy
donor, to which 250 µl of the plasma ultrafiltrate are added. If
this quantity is cytotoxic, the culture is repeated with 100 µl.
Since the clastogenic effects are related to oxyradical production, a culture
medium poor in free radical scavengers is recommended. TCM 199 or RPMI
1640 are convenient (5 ml per culture tube). The serum used for supplementation
(1 ml/culture) should not contain anti-oxidants from hemolysed erythrocytes.
Fetal calf serum is preferable to bovine serum, which may be rich in vitamin
E. Lymphocyte proliferation is stimulated by the addition of phytohemagglutinin
M or P. After 48 or 72 h of incubation at 37 ·C, the mitoses are
arrested in metaphase by the addition of colchicine 2 h before harvesting.
Microscopic slides are prepared for chromosomal analysis according to standard
procedures. The chromosomes of 50 well-spread and complete metaphases (10
on each of 5 coded slides) are examined for the presence of breaks, fragments,
exchanges, rings, dicentrics and other morphologically abnormal chromosomes.
Slides with a low mitotic index are eliminated, and the culture is repeated.
A series of ultrafiltrates is tested the same day on the cultures set up
with the blood of the same donor. Two additional control cultures without
ultrafiltrate serve for the establishment of the spontaneous chromosomal
aberration rate of the donor’s lymphocytes. This background level of aberrations
is subtracted from the aberration rate in ultrafiltrate-treated cultures
of the same blood donor. The difference between the two values is called
the adjusted clastogenic score (ACS). This way of treating of results is
necessary for comparison of clastogenic activity of samples collected at
subsequent dates and tested on cultures with different background level
of aberrations.
When background levels were studied on 10 parallel
cultures set up with the same blood, the variation did not exceed ±3
aberrations per 50 cells or ±6 aberrations per 100 cells studied.
This range was the same for two independent observers. Therefore a plasma
ultrafiltrate is considered to be clastogenic, if it induces more than
3 aberrations per 50 cells. In agreement herewith, the increase in aberrations
induced by ultrafiltrates from a series of 96 healthy blood donors did
not exceed 2 additional aberrations for the majority of them. Only with
5% of these normal samples, the increase represented +3, while no increases
of +4 or higher were observed (Emerit et al., 1995b).
Instead of chromosomal aberrations, other end-points
can be studied: sister chromatid exchanges, DNA strand breakage or mutations
at the HPRT locus (Emerit and Lahoud-Maghani 1989). However, CF do not
induce lesions in isolated DNA. They have to be studied on cellular systems
because of the indirect action mechanisms of break induction. CF may be
detected also by their superoxide stimulating properties using the cytochrome
C assay or chemiluminescence. However, the superoxide stimulating activity
is less stable than the clastogenic activity after freezing. One may also
use the appropriate biochemical assays for the various components, but
this would be time-consuming, more expensive and less sensitive. Indeed,
the different components may not always reach levels detectable with the
respective biochemical assays, while the clastogenic effects are the result
of the synergistic action of all CF components. Preliminary results indicate
correlations between CF activity, malondialdehyde levels, increased superoxide
production by phagocytes and diminished plasma thiol levels.
Diseases associated with oxidative stress and CF formation
1) Radiation exposure. CF are known since the early seventies, when
radiobiologists in Great Britain and the US reported chromosome-damaging
effects of plasma from therapeutically and accidentally irradiated persons
(Goh and Sumner, 1968, Hollowel and Littlefield, 1968). Further reports
came from A-bomb survivors in Hiroshima (Pant and Kamada, 1977). The existence
of radiation-induced CF was confirmed in our test system by the study of
plasma from adults and children exposed as a consequence of the Chernobyl
accident (Emerit et al., 1994, 1995b, 1997b). Compared to therapeutically
irradiated persons, the radiation doses received by this population are
relatively low. Longterm exposure may lead to CF formation as a consequence
of lipid peroxidation of cellular membranes and release of cytokines. Ionizing
radiation has been shown to increase TNF production by human peripheral
blood mononuclear cells in vitro (Krivenko et al., 1992). Low doses of
radiation resulted in increased release of TNF and of 13-hydroxy-octadecadienoic
acid by murine macrophages (Iwamoto and McBride, 1994). In our laboratory,
CF were isolated from the supernatant of irradiated cells after exposure
to only 50 cGy emitted by a 137 Caesium gamma source. The allowable cumulative
dose for Chernobyl accident recovery workers (liquidators) was 25 cGy.
CF formation did not occur, when the cells were irradiated in presence
of SOD (Emerit et al., 1994).
CF formation in irradiated persons is probably similar
to CF formation in chronic inflammatory diseases, a hypothesis supported
by the presence of inflammatory markers in irradiated individuals (Neriishi,
1991). This would explain why CF persist over many years after exposure:
more than 30 years in A-bomb survivors (Pant and Kamada, 1977) and more
than 10 years in liquidators (Emerit et al., 1994).
2) Chronic inflammatory diseases such as connective tissue disease,
ulcerative colitis, Crohn’s disease, hepatitis B and C, multiple sclerosis,
familial Mediterranean fever and others, are accompanied by CF formation.
Clastogenic activity is found not only in the plasma of patients, but also
in other body fluids and in the supernatants of cultures set up with whole
blood, isolated mononuclear cells or fibroblasts. Activated monocytes play
a major role in CF formation in chronic inflammatory diseases, such as
rheumatoid arthritis (Emerit et al., 1989). On the other hand, in familial
Mediterranean fever, a hereditary disease, in which paroxysmal attacks
of pain and fever are accompanied by a massive influx of neutrophils into
the serosal membranes, it could be shown that the spontaneously increased
superoxide production by neutrophils is correlated with the degree of clastogenic
activity in patients’ plasma (Sarkisian et al., 1997).
3) HIV-infection. There is more and more evidence for a role
of active oxygen species in HIV-infection and in the progress of the disease
to the acquired immune deficiency syndrome (AIDS). Exposure of latently
infected monocytes or CD4+ lymphocytes to oxidative stress was followed
by increased reverse transcriptase levels in the culture supernatants (Kalebic
et al., 1991). Clastogenic actvity was detected with our assay system in
plasma ultrafiltrates through 30,000 DA retentive for virus particles.
The plasma samples came from patients with AIDS, but also from asymptomatic
seropositive individuals, indicating that CF formation is an early event
in the disease (Fuchs and Emerit, 1995). Antiviral medication did not prevent
CF formation. There was a strong correlation between clastogenic scores
and decreases in the levels of plasma thiols and erythrocyte GSH. Anti-oxidant
vitamins, on the other hand, were in the normal range in patients with
highly clastogenic plasma. Clastogenic ultrafiltrates upregulated HIV-expression
in U1 cells, a chronically HIV infected promonocytic cell line. Exogenous
SOD inhibited the clastogenic and the virus-inducing effects of CF (Edeas
et al., 1997).
4) Psoriasis is a common skin disease, characterized by hyperproliferation
and incomplete differentiation of epidermal keratinocytes. Psoralen plus
UV-A (PUVA) is one of the treatments proposed for this disease. Since we
had previously reported tha PUVA-treated blood cultures show chromosomal
breakage due to formation of CF (Alaoui-Youssefi et al., 1994), we studied
plasma samples of 10 patients submitted to PUVA therapy. The clastogenic
activity of plasma ultrafiltrates increased significantly between the first
and the last (16th) exposure to PUVA. CF were present, to a minor degree,
before exposure to PUVA therapy. They were detected also in 14 out of 31
patients with psoriasis of similar severity, who were never exposed to
PUVA (Filipe et al., 1997). Superoxide production by inflammatory cells
is probably responsible for CF formation in this disease, in addition to
photosensitization reactions during PUVA. CF may contribute to the well-known
risk of photocarcinogenesis following PUVA therapy.
5) Ischemia-reperfusion. CF formation after ischemia-reperfusion
injury is probably also initiated by two sources of superoxide. Upon reoxygenation,
superoxide radicals are generated by the action of xanthine oxidase on
hypoxanthine, which is derived from progressive degradation of adenosine
triphosphate during ischemia (McCord, 1985). On the other hand, reperfusion
enhances neutrophil chemotaxis to the myocardium, and these activated cells
release superoxide and various mediators capable of promoting tissue injury.
CF could be isolated from the plasma of patients 5 min after opening of
the aortic clamp. As mentioned above, CF may stimulate the superoxide production
of phagocytic cells, and the ultrafiltrates from patients submitted to
ischemia-reperfusion had not only clastogenic, but also superoxide stimulating
properties on cells from healthy controls (Emerit et al., 1995a). Whether
superoxide production by neutrophils is the primary event, leading to CF
formation, or whether preformed CF activated neutrophils to produce superoxide
remains an open question. Once initiated, the vicious circle continues.
6) Congenital breakage syndromes. CF are regularly found in the
plasma of patients with ataxia telangiectasia, Bloom’s syndrome and Fanconi
anemia, and also in the supernatants of cell cultures set up with blood
or fibroblasts of these patients. While superoxide production by phagocytes,
lipid peroxidation and cytokine release appear to be involved in the other
above mentioned diseases, the reason(s) for the prooxidant state are less
evident in these syndromes. Fanconi anemia has been most intensively investigated.
Fanconi fibroblasts are particularly sensitive to hyperoxia (Saito et al.,
1993), and leukocytes exhibit an enhanced chemiluminescence response (Korkina
et al., 1992). Leukocyte DNA from homozygotes and heterozygotes shows oxyradical-
related base damage (Degan et al., 1995). In agreement herewith, CF were
found not only in plasma ultrafiltrates from homozygotes, but also from
heterozygotes, at the condition to concentrate the ultrafiltrate by a second
ultrafiltration step through a filter with a cut off at 1,000 daltons (Emerit
et al., 1995c). Spontaneous overproduction of TNF alpha in vitro and in
vivo has been reported (Roselli et al., 1994).
CF as an intermediate end-point for the evaluation of anti-oxidants in
clinical trials
Cellular and biochemical markers have been used as intermediate end-point
for the evaluation of the efficacy of a promising drug. The CF-test for
detection of superoxide-mediated clastogenesis represents a realiable and
sensitive assay for the presence of circulating pro-oxidants in a patient’s
plasma, which are detected due to their clastogenic properties. The fact
that activity is conserved over months in frozen samples is an advantage
in epidemiologic studies compared to cytogenetic studies of patients’ cells,
since it allows accumulation of many samples for study at a concenient
date.
SOD was regularly anticlastogenic in vitro in the
above mentioned diseases accompanied by CF, but we dispose only of rare
cases of SOD treatment in vivo. In 5 cases of rheumatoid arthritis, who
received intra-articular injections of bovine Cu-Zn SOD, CF were no longer
detectable after a 4-month treatment (Camus et al., 1980). In recent years,
the test was used for evaluation of prophylactic use of anti-oxidants in
high risk populations. For disease prevention, anti-oxidants in oral application
appeared preferable to SOD injections. With the authorization of the Armenian
Ministry of Health, thirty Armenian liquidators were treated with an extract
of Ginkgo biloba leaves (Tanakan, IPSEN Lab. Paris). The extract carrying
the number EGb 761 is standardized for a content of 24% Ginkgo flavone
glycosides and 8% Ginkgolides-Bilobalides (terpenes). A CF-test was performed
before the start of the treatment and at different intervals after arrest
of the treatment, which used the usual dose of 3×40 mg/day during
2 months. The clastogenic activity of the plasma was reduced to control
values when blood samples were taken in the first week arrest of the treatment.
The benefit of the treatment persisted more than 7 months and even up to
12 months in about one third of the liquidators. The fact that CF reappear
in the blood stream indicates that the process leading to CF formation
is not definitely halted by the treatment (Emerit et al., 1995b). In a
second study protective effects of a plant extract from soja, rice, wheat
germs, green tea and sesame (Anti-oxidant Biofactor A.O.B., AOA Company,
Kobe, Japan) could be demonstrated on another series of liquidators (Emerit
el al., 1997c). This extract contains various flavonoids, such as rutin,
daidzein, genistein etc., in combination with oligo-elements and small
quantities of vitamins. CF did not reach detectable levels, when control
samples were taken 6, 9 and 12 months after arrest of the 3-month treatment.
The workers experienced improvement of their general health and of their
working capacity. Both anti-oxidants will now be studied in a double blind,
placebo-controlled trial. The information that anti-oxidant treatment can
be disrupted during many months without reappearance of oxidative stress,
is important for cost evaluations in long-term intervention trials.
References
ALAOUI-YOUSSEFI, A., AROUTIOUNIAN, R., and EMERIT, I. (1994). “Chromosome
damage in PUVA-treated human lymphocytes is related to active oxygen species
and clastogenic factors.” Mutat. Res. 309:185–191.
AUCLAIR, C., GOUYETTE, A., LEVY, A., and EMERIT, I. (1994). “Clastogenic
inosine nucleotides as components of the chromosome breakage factor in
scleroderma patients.” Arch. Biochem. Biophys. 278:238–244.
CAMUS, J. P., EMERIT, I., MICHELSON, A. M., PRIER, A., KOEGER, A. C.,
and MERLET, C. (1980). “Superoxide dismutase et polyarthrite rhumatoide.”
Revue du Rhumatisme 47:489–492.
DEGAN, P., BONASSI, S., DE CATERINA, M., KORKINA, L., PINTO, L., SCOPASCASA,
F., ZATTERALE, A., CALZONE, R., and PAGANO, G. (1995). “In vivo accumulation
of 8-hydroxy-2’-deoxyguanosine in DNA correlates with release of reactive
oxygen species in Fanconi’s anemia families.” Carcinogenesis 16:735–742.
EDEAS, M., EMERIT, I., KHALFOUN, Y., LAZIZI, Y., CERNJAVSKI, L., LEVY,
A., and LINDENBAUM, A. (1997). “Clastogenic factors in plasma of HIV-infected
patients activate HIV-replication in vitro. Inhibition by superoxide dismutase.”
Free Radic. Biol. Med. 23:571–578.
EMERIT, I. (1990a). “Superoxide production by clastogenic factors.”
In: Free Radicals, Lipoproteins and Membrane Lipids (A. Crastes de Paulet,
L. Douste-Blazy, and R. Paoletti eds) Plenum Press New York, pp. 99–104.
EMERIT, I. (1990b). “Clastogenic factors: Detection and assay.” In:
Methods in Enzymology (L. Packer and A. N. Glazer, eds) Academic Press,
New York, pp. 555–564.
EMERIT, I. (1994). “Reactive oxygen species, chromosome mutation and
cancer: possible role of clastogenic factors in carcinogenesis.” Free Radic.
Biol. Med. 16:99–109.
EMERIT, I., and LAHOUD-MAGHANI, M. (1989). “Mutagenic effects of TPA-induced
clastogenic factor in Chinese hamster cells.” Mutat. Res. 214:97–104.
EMERIT, I., LEVY, A., and CAMUS, J. P. (1989). “Monocyte-derived clastogenic
factor in rheumatoid arthritis.” Free Radic. Biol. Med. 6:245–250.
EMERIT, I., KHAN, S. H., and ESTERBAUER, H. (1991). “Hydroxynonenal,
a component of clastogenic factors?” Free Radic. Biol. Med. 10:371–377.
EMERIT, I., LEVY, A., CERNJAVSKI, L., AROUTIOUNIAN, R., PANASSIAN, A.,
POGOSSIAN, A., MEJLUMIAN, H., SARKISIAN, T., GULKANDANIAN, M., QUASTEL,
M., GOLDSMITH, J., RIKLIS, E., KORDYSH, R., POLIAK, S., and MERKLIN, S.
(1994). “Transferable clastogenic activity in plasma from persons exposed
as salvage personnel of the Chernobyl reactor.” J. Cancer Res. Clin. Oncol.
120:558–561.
EMERIT, I., FABIANI, J. N., LEVY, A., PONZIO, O., CONTI, M., BRASME,
B., BIENVENU, P., and HATMI, M. (1965a). “Plasma from patients exposed
to ischemia reperfusion contains clastogenic factors and stimulates the
chemiluminescence response of normal leukocytes.” Free Radic. Biol. Med.
19:405–415.
EMERIT, I., OGANESSIAN, N., SARKISIAN, T., AROUTIOUNIAN, R., POGOSSIAN,
A., ASRIAN, K., LEVY, A., and CERNJAVSKI, L. (1995b). “Clastogenic factors
in the plasma of Chernobyl accident recovery workers: anticlastogenic effect
of Ginkgo biloba extract.” Radiation Res. 144:198–205.
EMERIT, I., LEVY, A., PAGANO, G., PINTO, L., CALZONE, R. and ZATTERALE,
A. (1995c). “Transferable clastogenic activity in plasma from patients
with Fanconi anemia.” Hum. Genet. 96:14–20.
EMERIT, I., GARBAN, F., VASSY, J., LEVY, A., FILIPE, P., and FREITAS,
P. (1996). “Superoxide-mediated clastogenesis and anticlastogenic effects
of exogenous superoxide dismutase.” Proc. Natl. Acad. Sci. USA 93:12799–12804.
EMERIT, I., QUASTEL, M., GOLDSMITH, J., MERKIN, L., LEVY, A., CERNJAVSKI,
L., ALAOUI-YOUSSEFI, A., POGOSSIAN, A., and RIKLIS, E. (1997a). “Clastogenic
factors in the plasma of children exposed at Chernobyl.” Mutat. Res. 373:47–54.
EMERIT, I., FILIPE, P., MEUNIER, P., AUCLAIR, C., FREITAS, J., DEROUSSENT,
A., GOUYETTE, A., and FERNANDES, A. (1997b). “Clastogenic activity in the
plasma of scleroderma patients: a biomarker of oxidative stress.” Dermatology
194:140–146.
EMERIT, I., OGANESIAN, N., AROUTIOUNIAN, R., POGOSSIAN, A., SARKISIAN,
T., CERNJAVSKI, L., LEVY, A., and FEINGOLD, J. (1997c). “Oxidative stress-related
clastogenic factors in plasma from Chernobyl liquidators: protective effects
of anti-oxidant plant phenols, vitamins and oligoelements.” Mutat. Res.
377:239–246.
FILIPE, P., EMERIT, I., ALAOUI-YOUSSEFI, A., LEVY, A., CERNJACSKI, L.,
FREITAS, J., and CIRNE DE CASTRO, J. L. (1997). “Oxyradical-mediated clastogenic
plasma factors in psoriasis. Increase in clastogenic activity after PUVA.”
Photochem. Photobiol. In press.
FUCHS, J., and EMERIT, I. (1995). “Clastogenic factors in plasma of
HIV-infected patients.” Free Radic Biol. Med. 19:843–848.
GOH, K. O., and SUMNER, K. (1968). “Breaks in normal human chromosomes.
Are they induced by a transferable substance in the plasma of irradiated
persons exposed to total body irradiation?” Radiation Res. 6:51–60.
HOLLOWELL, H. G., and LITTLEFIELD, L. G. (1968). “Chromosome damage
induced by plasma from irradiated patients. An indirect effect of X-ray.”
Proc. Soc. Exp. Biol. Med. 129:240–244.
IVAMOTO, K. S., and MCBRIDE, W. H. (1994). “Production of 13-hydroxy-octadecadienoic
acid and tumor necrosis factor alpha by murine peritoneal macrophages in
response to radiation.” Radiation Res. 139:103–108.
KRIVENKO, S., DRYK, S., KOMAROVSKAYA, M., and KARKANITSA, L. (1992).
“Ionizing radiation increases TNF/cachectin production in human peripheral
mononuclear cells in vitro.” Int. J. Hematol. 55:127–130.
MCCORD, J. M. (1985). “Oxygen-derived free radicals in post-ischemic
tissue injury.” N. Engl. J. Med. 312:159–162.
NERIISHI, K. (1991). “Possible involvement of a free radical mechanism
in late effects of A-bomb radiation.” Proc. 5th Int. Congr. on Oxygen Radicals;
Kyoto, Abstr.
PANT, G. S., and KAMADA, N. (1977). “Chromosome aberrations in normal
human leukocytes induced by the plasma of exposed individuals. Hiroshima
J. Med. Sci. 26:149–154.
SAITO, H., HAMMOND, A. T., and MOSES, R. E. (1993). “Hypersensitivity
to oxygen is a uniform and secondary defect in Fanconi anemia cells.” Mutat.
Res. 294:255–262.
SARKISIAN, T., EMERIT, I., ARUTYUNYAN, R., LEVY, A., CERNJAVSKI, L.,
and FILIPE, P. (1997). “Familial Mediterranian fever: clastogenic plasma
factors correlated with increased 02-production by neutrophils.” Hum. Genet.
In press.
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