Diverse xenotropic virus expressions in two NZB mouse breeding lines that differ in chromosome breakage.

Two breeding lines of the NZB/BI mouse strain, which was originated from Bielschowsky's stock and maintained as a conventional inbred colony since 1963, were developed by selective matings according to chromosome breakage incidence in bone marrow cells. These breeding lines were used because they represent an interesting experimental model, since they differ in the manifestations of the autoimmune process and the incidence of lymphomas. The present study showed that embryos from both breeding lines differed in the expression of the xenotropic type C RNA virus, characteristic of NZB mice. All 17 cultures initiated with embryos from the line showing high chromosome breakage (HB) had positive reverse transcriptase (RT) reactions after cocultivation with an indicator cell line (SIRC). On the contrary, only 3 of the 22 embryos from the line showing low chromosome breakage (LB) had RT activity at late passages (7-10). The chromosome studies done on these embryo cultures showed highly significant differences in the chromosome breakage incidence for the 2 breeding lines [10.6 and 20.5% of cells with aberrations for LB and HB embryos, respectively (P less than 0.001)].

Immunologic and enzymatic studies of two breeding lines of NZB mice that differ in chromosome breakage.

Two sublines of NZB/BI mice were developed by selective matings according to chromosome breakage frequencies. These sublines--HB, a line with high chromosome breakages, and LB, a line with low or normal breakage rates--were studied in regard to the age of the animals as it related to two different aspects. The first aspect was immunologic: A decreased response to T-cell mitogens was found in old NZB mice, but this response was more pronounced in HB mice. The response to the B-cell mitogen (lipopolysaccharide) was increased in both sublines as compared to that in BALB/c mice. The percentages of IgG-positive and theta-positive spleen cells were evaluated in both sublines: Some increase in IgG-positive cells was observed in the spleens of 2- to 8-month-old NZB mice and a slight decrease was seen after age 8 months. The percentage of theta-positive cells diminished according to the age of the mice, and the decrease occurred earlier in HB than in LB mice. The second aspect studied was enzymatic and concerned the levels of DNA alpha- and beta-polymerases and terminal DNA nucleotidyltransferase in the thymuses and spleens of these animals. The major finding noted was an augmentation of 100-200% in the terminal DNA nucleotidyltransferase levels in HB thymuses by comparison with LB thymuses. The levels of both polymerases were increased in spleen cells of HB mice as compared to those of LB mice.

Tumor incidence and development of autoimmune hemolytic anemia in two breeding lines of the NZB mouse strain that differ in chromosome breakage.

Increased chromosome breakage observed in NZB mice was studied. Breeding experiments with mice selected according to breakage frequencies provided evidence that the proportion of mice with high breakage (HB) and low breakage (LB) figures in the progeny depends on the phenotype of the parents. Selective breeding for the "chromosome breakage" characteristic was successful and resulted in the separation of a breeding line with LB incidence. However, the selection could not be continued beyond the fourth generation for the mice with HB incidence because of lethal factors. Comparative studies of HB mice from the HB line and LB mice from the LB line showed significant differences for tumor incidence and positivity of the Coombs' test.

Reactive oxygen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis.

Superoxide radicals may induce genotoxic effects by indirect action mechanisms, implicating the formation of more long-lived, secondary clastogenic material called chromosome breakage factors or clastogenic factors (CF). CF are produced via the intermediacy of superoxide, and stimulate further superoxide production by competent cells. This results in a selfsustaining and longlasting process of clastogenesis, which may exceed the DNA repair system and ultimately lead to cancer. An increased cancer risk is indeed observed in conditions accompanied by CF formation. These include irradiated persons, asbestos workers, patients with chronic inflammatory diseases, HIV-infected persons, and the congenital breakage syndromes ataxia telangiectasia, Bloom's syndrome, and Fanconi's anemia. Because reactive oxygen species (ROS) are implicated in CF formation and CF action, antioxidants may be protective as anticlastogens and consequently as anticarcinogens. In persons at high risk because of their occupation, life style or place of residence, the presence of CF may represent an indication for chemoprevention of cancer by antioxidants. CF can be useful as biochemical markers and intermediate endpoints for the evaluation of promising drugs. They are therefore not only of interest as a mechanism by which ROS may exert genotoxic effects, but also have practical implications.

Superoxide and hydrogen peroxide production by macrophages of New Zealand black mice.

Resident peritoneal macrophages from New Zealand Black (NZB) mice release O2- and H2O2 after adherence to a plastic surface without any chemical or particulate stimulant. This phenomenon is age dependent and more pronounced in animals with sever autoimmune disease. Significant differences were observed between the high and low breakage NZB sublines (HB and LB), which were previously developed by selective matings on the basis of chromosome breakage rates. The LB subline differs significantly from the HB subline with respect to autoimmune hemolytic anemia and tumor incidence. When the macrophages were stimulated with the tumor promoter TPA, the number of "responders" was higher in the HB than in the LB subline and correlated with the degree of splenomegaly, that is, with the severity of the disease. A negative response to agonist stimulation and very low spontaneous production of active oxygen species was observed in NZW and Swiss mice, which is the normal finding for resident macrophages according to data from the literature. The increased superoxide and hydrogen peroxide production by macrophages of NZB mice is discussed with respect to autoimmune disease and cancer.

Hydroxynonenal, a component of clastogenic factors?

Exposure of human lymphocyte cultures to superoxide generated by the xanthine-xanthine oxidase (X-XO) system, resulted in formation of a clastogenic factor (CF), as expected from previous work. We speculated that arachidonic acid (AA), the major polyunsaturated fatty acid of biological membranes, was oxidized via the cyclooxygenase-lipoxygenase pathways or nonenzymatically by oxygen free radicals in the culture medium to products with clastogenic properties. In the present study, we analyzed CF for AA-derived products and tested corresponding commercial standards for their clastogenic properties. The results show that prostaglandins, thromboxane, and H(P)ETEs were not increased in supernatants from X-XO treated cultures compared to untreated cultures. Synthetic H(P)ETEs added to the medium of lymphocyte cultures were only slightly or not clastogenic. In contrast hereto, the degradation product 4-hydroxynonenal was found in 50% of CF samples, while it was absent in all 43 control samples. The kinetics of detectability in the culture medium was similar to that of CF. Also, the clastogenic effect of synthetic 4-hydroxynonenal at concentrations as low as 0.1 microM suggested that this aldehyde, known for its genotoxic effects, was a clastogenic component of CF. The indirect action mechanisms of 4-hydroxynonenal via inactivation of functional SH groups in DNA polymerases, may explain why chromatid-type damage is predominant in lymphocytes exposed to CF in the Go-G1 phase of the cell cycle. This particularly was already stressed 20 years ago in the first observations of radiation-induced CF. However, 4-hydroxynonenal is not the only clastogenic component of CF.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocyte-derived clastogenic factor in rheumatoid arthritis.

Blood or lymphocyte cultures from patients with rheumatoid arthritis show increased chromosome breakage. This is due to the presence of a clastogenic factor (CF) inducing also chromosome damage in blood cultures of healthy persons. CF may be isolated not only from patients' plasma or synovial fluid, but also from the supernatant of blood or lymphocyte cultures. No CF was detectable, if the lymphocyte cultures were free of other contaminating blood cells. Addition of neutrophils did not considerably influence the production of CF, and platelets were without any effect. However, addition of increasing numbers of monocytes resulted in increasing clastogenic activity. Also monocytes in adherence, in absence of lymphocytes and without any chemical stimulant, produced CF. This indicates that monocytes are responsible for CF production. The protective effect of superoxide dismutase, as well against CF formation as against CF action on cells of normal subjects, suggests a role of the superoxide radical O2-. Inhibitors of arachidonic acid metabolism were only slightly anticlastogenic.

Radiation-induced clastogenic factors: anticlastogenic effect of Ginkgo biloba extract.

Clastogenic factors (CFs) were first described in the blood of persons irradiated accidentally or for therapeutic reasons. Work of our laboratory has shown that they occur also under other circumstances, which are characterized by oxidative stress, and that CF-induced chromosome damage is regularly prevented by superoxide dismutase (SOD). Recently we found CFs in a high percentage of salvage personnel of the Chernobyl reactor accident. These liquidators represent a high-risk population and might benefit from cancer chemoprevention by antioxidants. SOD would have to be injected and is not appropriate for long-term prophylactic treatment. In the present study, we therefore evaluated the anticlastogenic effect of the Ginkgo biloba extract EGb 761, which is known for its superoxide scavenging properties. EGb 761 was tested on CF-treated blood cultures of healthy donors. After establishing the optimal protective EGb concentration, using CFs produced by irradiation of whole blood from healthy volunteers, the extract was tested on cultures exposed to CFs from plasma of persons irradiated as liquidators. The anticlastogenic effect could be confirmed for a final concentration of 100 micrograms/ml. In 12 consecutive experiments, CFs induced an average of 18.00 +/- 4.41 aberrations/100 cells. This was reduced to 7.33 +/- 3.08 in the parallel cultures receiving 100 micrograms/ml EGb 761 (p < .001). SOD was anticlastogenic in the same system at concentrations of 30 cytochrome C units/ml (approximately 10 micrograms/ml). Preliminary results obtained in a small series of liquidators showed regression or complete disappearance of CFs in the plasma after 2 months of treatment with EGb 761 (3 x 40 mg/d).

Clastogenic factors in plasma of HIV-1 infected patients activate HIV-1 replication in vitro: inhibition by superoxide dismutase.

The frequent neoplastic disorders present in HIV-infected patients and the implication of oxidative stress in AIDS-Kaposi's sarcoma pathogenesis prompted us to study whether the mechanisms implicated in genotoxic effects of clastogenic factors (CFs) (i.e., chromosome damaging materials released by cells under conditions of oxidant stress) can play a role in HIV-1 expression and whether exogenous superoxide dismutase can inhibit the clastogenic and HIV-inducing effects of CFs. CFs were found in the plasma of all HIV-1 infected patients (n = 21) of this study group, in asymptomatic (CDC II) as well as in symptomatic patients (CDC IV). In addition to their chromosome damaging effect, CFs are able to upregulate HIV-1 expression in U1 cells and in PBMCs activated with PHA and IL2 at all time points (p < .05). Their formation, therefore, is an early event in the disease. It occured despite antiviral medication in these patients. Superoxide dismutase inhibited the clastogenic and the viral inducing effects (p < .05). On the basis of our findings, association of SOD mimetics or superoxide scavengers with antiviral drugs may be a new therapeutic approach. This polytherapy, if started early enough after infection, may prolong the latency period and limit the emergence of drug-resistant viral strains.

Oxyradical-mediated chromosome damage in patients with familial Mediterranean fever.

Increased chromosome breakage is observed in patients with familial mediterranean fever (FMF). Their plasma contains clastogenic material inducing chromosome damage in cells from healthy persons. It is proposed that increased oxyradical generation by activated polymorphonuclear cells in blood and serosal fluids of these patients leads to the formation of a clastogenic factor (CF), as it is observed in other chronic inflammatory diseases. Also similar to these diseases, the clastogenic effects are prevented by superoxide dismutase and partially by inhibitors of arachidonic acid metabolism.

Clastogenic factors in plasma of HIV-1 infected patients.

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-1 infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (< 10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 +/- 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 +/- 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 +/- 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 +/- 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.

Plasma from patients exposed to ischemia reperfusion contains clastogenic factors and stimulates the chemiluminescence response of normal leukocytes.

Clastogenic factors (CFs) are released by cells exposed to superoxide radicals and are found in various situations of oxidative stress. Certain of their components stimulate further superoxide production by competent cells, as shown with cytochrome c assay in previous work. In the present study, we report CF formation after ischemia reperfusion in patients undergoing coronary bypass surgery. Plasma ultrafiltrates, collected 20 min after reperfusion, had clastogenic properties in contrast to those collected before ischemia. We also show that the luminol-enhanced chemiluminescence response of neutrophils from healthy persons is increased when these cells are exposed to CF-containing postreperfusion samples from patients. Light emission was reduced to control values in the presence of superoxide dismutase. The burst of oxyradicals upon reperfusion is probably the initiating event of CF formation, which in turn leads to further oxyradical generation. This amplification process may explain why detectable levels of CF need a delay of at least 10 min. The activated state of neutrophils in ischemia reperfusion is at once a consequence and a source of CFs. Individual variation in the persistence of this clastogenic and leukocyte-activating material was observed. Therefore, antioxidants for prevention of ischemia reperfusion injury should be continued during the postoperative course.

Free radicals and aging of the skin.

Cutaneous aging is the result of genetically determined or intrinsic aging superimposed by degenerative changes due to actinic irradiation, also called photoaging. The manifestations of cutaneous aging, as it relates to the perception of age, is caused by ultraviolet light, in particular in those parts of the body exposed daily to solar radiation. Free radical generation in the skin by UV light and from other sources, such as cellular infiltrations or the xanthine oxidase reaction, may be detected by direct and indirect methods. The decrease in antioxidant enzymes and small molecular weight antioxidants such as glutathione, vitamin E and ubiquinone upon exposure to UV light is an indication that the pro-antioxidant balance can be overwhelmed by acute or chronic photo-oxidative stress. Antioxidant supplementation is therefore a means for prevention or at least retardation of premature cutaneous aging.

Active oxygen species, articular inflammation and cartilage damage.

Rheumatoid arthritis and osteoarthritis are age-related diseases, in which degenerative changes (arthrosis) and superimposed inflammatory reactions (arthritis) lead to progressive destruction of the joints. Active oxygen species derived from various sources play a role in this process, which may be influenced by appropriate treatment with antioxidants and free radical scavengers.

Lessons learned from the study of immigrants to Israel from areas of Russia, Belarus, and Ukraine contaminated by the Chernobyl accident.

During the past 6 years, immigration to Israel of 700,000 persons from the former Soviet Union (FSU) included about 140,000 from radiocontaminated regions of Belarus, Ukraine, and Russia near Chernobyl. In Beer Sheva, a major center for immigrant absorption in Israel, a primary objective was to evaluate their health status and to refer them for care. 137Cs levels in 1228 men, women, and children were measured with a portable whole-body counter. Whole-body counts showed clear correlation with the degree of 137Cs ground contamination in previous regions of residence. The population could thus be sub-divided according to degree of exposure, based on previous regions of residence. The thyroid status of 300 local immigrant children was evaluated because of the increased risk of childhood thyroid cancer in the regions from which they came. This group was subdivided into comparative groups of children who came from less and more contaminated areas according to the International Atomic Energy Agency soil 137Cs contamination maps. Enlarged thyroids were found in about 40% of both groups. One 12-year-old girl from Gomel had a malignant papillary carcinoma. Thyroid-stimulating hormone levels, though within normal limits, were significantly greater (p < 0.02) for girls from high exposure regions. Liquidators showed significant increases in serum clastogenic factor and in the number of circulating glycophorin A-mutated red cells. In studies of over 700 people from both radiocontaminated and unaffected regions of the FSU, evidence for posttraumatic stress disorder was found more frequently in persons coming from the more contaminated areas.

Transferable clastogenic activity in plasma from persons exposed as salvage personnel of the Chernobyl reactor.

Clastogenic factors were first described in the plasma of people who had been accidentally or therapeutically irradiated. They were found also in A-bomb survivors, where they persisted for many years after the irradiation. The present study searched for these factors in the plasma of 32 civil workers from Armenia, who had been engaged as "liquidators" around the Chernobyl atomic power station in 1986. It also included 15 liquidators who had emigrated from the ex-Soviet Union to Israel. Reference plasma samples were obtained from 41 blood donors from the Armenian Blood Center in Yerevan. The samples were tested for their clastogenic activity in blood cultures from healthy donors. The majority of results from the liquidators exceeded those from the unexposed reference samples. The samples from the first Armenian group, with the higher average irradiation dose (0.6 +/- 0.6 Gy), were more clastogenic than those from the second group exposed to 0.2 +/- 0.2 Gy. The number of aberrations in the test cultures was 17.9 +/- 2.9% and 10.5 +/- 3.8% respectively, compared to 5.7 +/- 3.2% in the cultures exposed to the reference ultrafiltrates from Armenian blood donors. The samples from the Israeli liquidators also induced significantly increased aberration rates (14.0 +/- 3.9% aberrant cells). The clastogenic activity was regularly inhibited by superoxide dismutase, indicating that the chromosome-damaging effects of radiation-induced clastogenic factors are exerted via the intermediation of superoxide radicals, as is known for clastogenic factors of different origin.

Clastogenic factors in the plasma of Chernobyl accident recovery workers: anticlastogenic effect of Ginkgo biloba extract.

Clastogenic factors are found in the plasma of persons irradiated accidentally or therapeutically. They persisted in the plasma of A-bomb survivors over 30 years. Clastogenic factors were found in 33 of 47 Chernobyl accident recovery workers (often referred to as liquidators) in a previous study (I. Emerit et al., J. Cancer Res. Clin. Oncol. 120, 558-561, 1994). In the present study, we show that there is a positive correlation between clastogenic activity and dose and that these biomarkers of oxidative stress can be influenced successfully by appropriate antioxidant treatment. With the authorization of the Armenian Ministry of Health, 30 workers were treated with antioxidants from Ginkgo biloba leaves. The extract EGb 761 containing flavonoids and terpenoids was given at a daily dose of 3 x 40 mg (Tanakan, IPSEN, France) during 2 months. The clastogenic activity of the plasma was reduced to control levels on the first day after the end of the treatment. A 1-year follow-up showed that the benefit of the treatment persisted for at least 7 months. One-third of the workers again had clastogenic factors after 1 year, demonstrating that the process which produced clastogenic factors continued. However, the observation that antioxidants do not have to be given continuously is encouraging for intervention trials on a large-scale basis. These appear justified, since clastogenic factors are thought to be risk factors for the development of late effects of irradiation.

Clastogenic factor in ischemia-reperfusion injury during open-heart surgery: protective effect of allopurinol.

The hypothesis tested was that free radicals generated following ischemia and reperfusion in cardiac operations can produce clastogenic factor that results in chromosomal aberration. Fourteen randomized patients undergoing coronary artery bypass grafting were divided into two groups. In Group 1 (7 patients), myocardial protection was achieved using a cardioplegic solution without allopurinol. In Group 2 (7 patients), 100 mg of allopurinol (xanthine oxidase inhibitor) was added to the solution. In both groups, blood samples were taken from the coronary sinus before the aorta was clamped and 20 minutes after myocardial reperfusion was achieved. The blood samples were used to study the patients' chromosomes. The results were given as the percentage of chromosomal aberrations observed in 100 mitoses. There were no significant differences between the preischemic values in both groups and the postischemic values in Group 2. On the other hand, there was a significant difference between the postischemic values in Groups 1 and 2 (p less than 0.01). In conclusion, reperfusion following myocardial ischemia in cardiac operations can produce clastogenic aberrations. This clastogenic activity can be reduced by adding allopurinol to the cardioplegic solution.

Protective effect of superoxide dismutase against hair graying in a mouse model.

Oxygen free radicals play a role in the aging process, and the protective effect of various antioxidants has been intensively studied, in particular for cutaneous aging. Besides hereditary factors, free radical-mediated damage to melanocytes of the hair follicle has been considered as a mechanism for aging of the hair. It was the aim of this study to evaluate the role of photosensitization reactions for hair graying and to demonstrate potential protective effects of superoxide dismutase (SOD). Mice with black hair were depilated with the fingertips on a surface of 6 x 2.5 cm on both sides of the dorsum. The right side received five applications of a SOD-containing gel before exposure to psoralen (concentration 0.5 mg/mL) plus UV-A (365 nm, 4 J/cm2). The left side was pretreated in the same way with a gel free of SOD. When the hair started growing again, the SOD-protected side was covered with black hair, whereas the hair on the vehicle-treated side was gray or white in 27 of the 30 animals studied. The 0.01% SOD concentration was as protective as the 0.1% concentration. Heat-inactivated SOD, applied in another five animals, was not protective. Using fluorescent labeling of the SOD with fluorescein isothiocyanate, epifluorescence microscopy and digital imaging processing, we show that SOD applied to the skin surface penetrates through the follicular appendages, as well as through the unbroken stratum corneum. Our findings suggest that superoxide radicals, generated by interaction of UV-A light with the sensitizer, initiated the formation of secondary products with well-known DNA-damaging effects, such as lipid peroxidation products and tumor necrosis factor alpha. SOD prevented the damage to melanocyte DNA by dismutating superoxide. Photosensitization may be another mechanism for hair graying, which can be influenced by antioxidants. Given the large number of exogenous and endogenous sensitizers, this mechanism deserves further study for human hair graying.

Oxyradical-mediated clastogenic plasma factors in psoriasis: increase in clastogenic activity after PUVA.

Psoriasis is a common skin disorder characterized by hyperproliferation and incomplete differentiation of epidermal keratinocytes. Psoralen plus UVA (PUVA) is one of the treatments proposed for this disease. We had reported previously that exposure of regular blood cultures from healthy donors to PUVA leads to chromosomal breakage via the formation of transferable clastogenic materials, a phenomenon inhibitable by superoxide dismutase. In the present paper we show that these clastogenic factors (CF) are also formed in vivo. The CF were found in about 50% of the psoriasis patients studied (14 out of 31). In PUVA-treated psoriasis patients, the clastogenic activity of the plasma increased significantly between the first and the last (16th) exposure to PUVA. We hypothesize that CF formation in psoriasis is similar to that in other diseases accompanied by oxidative stress, in particular chronic inflammatory diseases with autoimmune reactions such as lupus erythematosus, progressive systemic sclerosis, rheumatoid arthritis and others. Increased superoxide production by phagocytes, formation of lipid peroxidation products and release of cytokines are considered to be responsible for the superoxide-stimulating and chromosome-damaging properties of patients' plasma. During PUVA therapy, superoxide generated via the interaction of psoralen with UVA may contribute to CF formation in addition to superoxide from inflammatory cells. An increased risk of cancer and leukemia is observed in diseases accompanied by CF formation. Therefore CF may contribute to the well-known risk of photocarcinogenesis by PUVA therapy. This additional risk may be preventable by antioxidants and superoxide scavengers.