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.

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