Clastogenic factors in plasma of HIV-1 infected patients

Free Radical Biology & Medicine, Vol. 19, No. 6, pp. 843-848, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in the USA. All rights reserved

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Brief Communication CLASTOGENIC

JURGEN

FUCHS,*

FACTORS

INGRID

IN PLASMA

EMERIT,’

ARLETTE and RAINER

*Department

of Dermatology,

(Received

OF HIV-l

LEVY,’

LIDIA

INFECTED

CERNAJVSKI,+ HELMUT SCH&ER,*

MILBBADT*

Frankfurt University Hospital, Germany; and ‘Institut Universite Pierre et Marie Curie, Paris, France

15 December

1994; Revised

PATIENTS

15 March 1995; Accepted

Biomedical

des Cordeliers,

22 March 1995)

Abstract-The

objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-l infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (
factors,

Free radicals,

Oxidants,

Antioxidants,

antioxidants ascorbate, lo glutathione and N-acetylcysteine, “3” and lipoate.” It was suggested that the shift in the redox equilibrium toward an oxidizing state contributes to the pathogenesis of the disease (e.g., participating in the self-perpetuation of viral replication, immunodysregulation, and carcinogenesis) .14-” The cellular antioxidant status was suggested to be an important factor for determining the latency period of HIV infection.16 In the present study, we investigated the presence of clastogenic factors in the plasma of HIV-infected persons. Clastogenic factors have been recognized for more than 20 years as an indirect effect of ionizing radiation. Because of their persistence in the blood of irradiated persons many years after exposure, they have been considered as risk factors for late effects of ionizing irradiation, such as cancer and leukemia.‘7 Previous work of our laboratory has shown that clastogenic fac-

INTRODUCTION

Experimental evidence is accumulating that HIV-l infected patients are in oxidative imbalance, which starts early in the course of the disease. Low levels of serum and tissue antioxidants and elevated concentrations of peroxidation products are reported.‘-’ It was also shown that DNA damage secondary to photosensitization reactions and singlet oxygen formation promotes virus reactivation and that exposure of latently HIVinfected monocytes or CD4+ lymphocytes to hydrogen peroxide-mediated oxidative stress is followed by increased reverse transcriptase activity in the culture supernatants.’ On the other hand, virus expression in chronically infected cells could be suppressed by the

Address correspondence Aschaffenburg, Germany.

to: Jtirgen Fuchs, Heinsestrasse

HIV infection

8, 63739

843

J. FUCHS et al.

844

tors are found in a variety of diseases and that their formation and their clastogenic action are related to increased oxygen free radical production. Chronic inflammatory diseases with autoimmune reactions, as well as the congenital breakage syndromes Ataxia telangiectasia, Bloom’s syndrome, and Fanconi anemia, are accompanied by clastogenic factors.” The strongest argument for the implication of superoxide anion radicals in clastogenic factor formation and action came from in vitro models, in which cell cultures were exposed to a source of superoxide anion. Clastogenic factor formation in these cultures, as well as the clastogenie action of preformed clastogenic factor in other cell cultures, could be prevented by superoxide dismutase. ‘y.20Using the cytochrome c assay, it was shown that clastogenic factors stimulate superoxide production by neutrophils and monocytes.2’ The clastogenic factors isolated to date from patient sera or from cell culture supernatants share certain characteristics, including low molecular weight, as they are able to pass through a lO,OOO-dalton filter.22 The cellular origin of clastogenic factors is indicated by the fact that their formation is only demonstrable in the presence of cells, but not after exposure of cellfree medium to oxygen radicals. Thiobarbituric acid reactive substances, conjugated dienes, and the aldehyde 4-hydroxynonenal have been isolaled from clastogenic factor preparations, suggesting an association between clastogenic factor formation and membrane lipid peroxidation. In addition, inosine nucleotides (ITP and IDP), which are primers of superoxide production by phagocytes and also compete with the adenosine 5 ‘&phosphate ( ATP) binding sites of DNA topoisomerases, have been detected in clastogenic factor preparations of scleroderma patients.24 Tumor necrosis factor alpha has been found in clastogenic factor preparations from patients with Fanconi anemia.25 Formed via the intermediacy of superoxide and stimulating further superoxide production by competent cells, clastogenie factors are self-sustaining and can be considered as biomarkers of oxidative stress. Their presence in the plasma of HIV-infected persons, as demonstrated by the following data, is another argument in favor of the role of oxygen free radicals in HIV infection. MATERIALS

AND METHODS

Patients Out- and in-patients from the Department of Dermatology, University Hospital, Frankfurt, Germany, were included in the study if they were HIV-l seropositive, older than 18 years, and male. The exclusion criterion was intake of minerals, vitamins, or other antioxidants. A nutritional questionnaire was taken from each pa-

tient, and all patients were in a satisfactory nutritional state. A total of 22 patients were included, and written consent to participate in the study was provided by each individual. The patients were divided into two groups according to the number of CD4+ cells. Group A comprised nine patients with more than 200 cells/ ml, and group B contained 13 patients with less than 200 cells/ml blood. Plasma antioxidants and lipid peroxidation products were analyzed in these patients and published earlier.6

Reference

plasma

Plasma samples from 20 healthy HIV-negative blood suppliers of similar age, recruited among the blood donors of the Centre de Transfusion in Paris, were studied in comparison to the plasma from HIVinfected patients.

Sample preparation For clastogenic factor detection, the plasma was ultrafiltered through a filter with a cutoff at 10,000 daltons (Amicon YM 10, Danvers, MA), according to procedures reported in detail elsewhere, and stored on liquid nitrogen.2’ The ultrafiltrates were delivered frozen to Paris, where they were tested for their clastogenie activity in a cytogenetic test system. The reference samples were frozen also and handled in the same way as the patients samples.

Cytogenetic

assay

Aliquots of plasma ultrafiltrates were added at the beginning of the cultivation period to the test cultures, which were set up with 0.5 ml of whole blood from healthy donors in 5 ml tissue culture medium (TCM 199 from Flow Lab, Paris, France) and 1 ml fetal calf serum (Gibco, Paris, France). Lymphocyte proliferation was stimulated by addition of phytohemagglutinin (Wellcome Diagnostics, Dartford, UK). After 72 h of incubation at 37”C, the mitoses were arrested in metaphase by addition of colchicine 2 h before harvesting. Microscopic slides were prepared for chromosomal analysis according to standard procedures. The chromosomes were examined on coded slides for the presence of gaps, breaks, fragments, exchanges, rings, and dicentrics by experienced cytogeneticists. A minimum of 50 mitoses was studied. The total of chromosomal aberrations was compared between cultures exposed to ultrafiltrates (HIV patient’s samples or reference samples) and untreated controls set up with blood from the same donor. In several experiments, the culture had to be repeated with a smaller quantity of ultra-

Clastogenic

factors

=m

$

in HIV-I

845

patients

p<

0.01

30

B

T-r-

0

HIVAB+SOD

Fig. 1. Chromosomal aberrations in lymphocytes of healthy blood donors induced by plasma ultrafiltrates from healthy controls and HIV-l infected patients, group A (T-helper cells > 200/~1), group B (T-helper cells < 200/~1). Inhibition of clastogenic activity by addition of SOD (30 cytochrome c units/ml final concentration).

filtrate ( 100 ~1 instead of 250 ~1) because of cytotoxic effects, resulting in a reduced mitotic index. The influence of superoxide dismutase (SOD) was studied in 16 of the 22 samples. The antioxidant enzyme was added to the cultures at the beginning of the 72-h cultivation period at a final concentration of 30 cytochrome c units/ml. The statistical analysis was done with the Mann-Whitney test.

RESULTS

The mean chromosomal aberration rate of the untreated test cultures set up with the blood from seven healthy donors was 7.4 k 2.5 per 100 cells and slightly, but not significantly, higher than the laboratory standard (4.0 + 2.6 per 100 cells, range 0 to 10) under the given culture conditions. The aberration rates induced by ultrafiltrates from patients of group A in the test cultures set up with blood of healthy persons was 18.0 5 4.0 per 100 cells, and that of group B was 22.3 5 7.9. The ultrafiltrates from patients group A + B induced a mean chromosomal aberration rate of 20.5 f 6.8 per 100 cells. In contrast, the mean aberration rate induced in test cultures by ultrafiltrates from 20 reference plasma samples (healthy HIV-negative blood donors) was 6.3 + 2.9 per 100 cells (Fig. 1). Compared to untreated cultures, this represents an increase of 14.2 chromosomal aberrations per 100 cells for the ultrafiltrates from patients (p < .Ol) and of 1.4 chromosomal aberrations per 100 cells only for reference plasma ultrafiltrates. The aberration rates induced by ultrafiltrates from patients of group A were lower than those observed in the cultures exposed to ultrafiltrates from group B, but the difference did not reach statistical significance (Fig. 1, Table 1) . The chromosomal aberrations consisted principally of chro-

matid-type aberrations, as is usual for clastogenic factor induced chromosome damage. If gaps were excluded from the total, the aberration rate was reduced to 13.4 aberrations per 100 cells, but it remained statistically different from the controls (aberration rate 4.7 per 100 cells after exclusion of gaps). If SOD was added 30 min before exposure of cells to the HIV patient ultrafiltrates, the aberration rates were regularly lower in the treated cultures ( 12.0 + 4.0 per 100 cells, p < .05; Fig. 1). As shown in Table 2, patients were subdivided into two groups according to the clastogenic activity in their plasma. When the 10 patients with low clastogenic activity ( 12 to 18 aberrations per 100 cells) were com-

Table 1. Frequency and Nature of Chromosomal Aberrations in Test Cultures Exposed to Plasma Ultrafiltrates of HIV-Infected Patients or Healthy Subjects

Number of analyzed metaphases Number of aberrations Number of aberrations/per 100 cells Group A and B Range Group A (n = 9) Range Group B (n = 13) Range Background of aberrations in test cultures Gaps of one or both chromatids Breaks of one chromatid Breaks of both chromatids Fragments, dots Dicentrics Total

HIV (n = 22)

Healthy Subjects (n = 20)

1100 225

1000 63 6.3 2 2.9

20.0 + 6.8 12-39 18.0 t 4.0 12-26 22.3 2 7.9 14-39 7.4 2 2.5 34.70% 41.70% 15.60% 5.80% 2.20% 100.00%

2-10

4.9 ? 1.8 31.70% 60.30% 7.90% 0% 0% 99.90%

J. FUCHS et al.

846 Table 2. Comparison

of Clastogenic

Activity

Low Breakage (l2-18%) Malondialdehyde 4-Hydroxynonenal Total glutathione Total plasma thiols Glutathione peroxidase Selenium Zinc Beta carotene Vitamin A Vitamin C Vitamin E Data antioxidants

0.336 0.194 2.38 82.16 148.00 61.25 71.5 97.20 1.93 8.25 25.49

and peroxidation

2 2 2 -+ 2 + 2 5 ? 2 *

and Plasma

n(l0)

0.247 0.073 2.42 36.72 39.56 17.86 8.35 39.58 0.89 3.30 7.77

products

6 6 6 6 4 4 5 I 8 7

Levels of Peroxidation High Breakage (20-39%) 0.678 0.185 1.75 71.6 138.00 48.80 67.00 76.11 1.93 7.35 28.10

k 2 ? k k 2 5 -f t +k

0.505 0.071 1.85 37.49 23.24 15.06 21.87 27.12 0.53 4.99 8.03

Products

n(l2) 7 7 5 10 5 5 9 9 9 10 9

and Antioxidants

Normal

Range

0.18-0.55 hmolil 0.05-O. 15 pmolll 5.66-8.90 pmol/l IO& 136 PrnoUl 130- 190 un 50- 100 pg/l 73-127 &l > 47 /&g/l 1.1-2.8 pmol/l 8.0- 12.0 mg/l 11.8-46.4 PrnoUl

from ref. 6.

pared to the group of 12 patients with high clastogenic activity (between 20 and 39 aberrations per 100 cells), differences were observed with respect to lipid peroxidation, since the high breakage group showed higher values for malondialdehyde. The aldehyde 4-hydroxynonenal was equally increased in both groups. Furthermore, the high breakage group showed a tendency toward lower concentrations of plasma antioxidants. Glutathione levels, as well as total plasma thiols, were decreased considerably in both groups, but more in the high-breakage group. Also, glutathione peroxidase (GPX) showed lower activity in the high-breakage group but remained in the normal range. Vitamin C, selenium, and zinc were beyond the lower limit of normal, and again lower in the group with the high breakage rates. No differences between both groups were noted for the antioxidant vitamins A and E, which were in the normal range. Beta carotene was also in the normal range, but lower in the high-breakage group.6 All 22 patients were in a satisfactory nutritional state. Statistically, however, all differences in lipid peroxidation products and plasma antioxidants between the low- and high-breakage groups did not reach significance.

DISCUSSION

While plasma ultrafiltrates from HIV-negative, healthy subjects did not significantly increase the number of chromosomal aberrations in the test cultures set up with blood of other healthy donors, the plasma ultrafiltrates from HIV- 1 infected patients were clearly clastogenic. We conclude that the plasma of these patients contains clastogenic substances, comparable to the clastogenic factors described in other pathological conditions accompanied by oxidative stress. The clastogenic activity was found in the same plasma fraction, since the same ultrafiltration membrane was used (cut-

off at 10,000 daltons). Among the identified clastogenie components of clastogenic factors mentioned earlier, we can confirm the presence of the aldehyde 4-hydroxynonenal in plasma of HIV-infected patients. Although 4-hydroxynonenal concentration was increased in the plasma of HIV patients, there was no difference between the patients with moderate clastogenie activity and those with high clastogenic activity, indicating that the clastogenic effect was also due to other agents. The two other identified components of clastogenic factors (inosine triphosphate = ITP, and tumor necrosis factor alpha = TNF alpha) were not yet studied. However, it is known from the literature that blood monocytes from HIV-infected patients spontaneously produce high levels of this cytokine.26 Soluble TNF alpha is a peptide with a molecular weight of 17,000, and one may wonder whether it can pass through filter YMlO used for the ultrafiltration of patient’s plasma. This is indeed the case for about 25% of the total, as shown previously by comparison of TNF alpha concentrations in plasma and ultrafiltrates from Fanconi patients (Roselli F, Institut Curie Paris, personal communication). The identification of TNF alpha as the main component of the clastogenic material found in HIV patients awaits confirmation. Clastogenic factors have be considered as risk factors for the occurence of late effects after exposure to ionizing irradiation.17 In agreement herewith, a high risk of cancer and leukemia is observed in association with clastogenic factors in ataxia telangiectasia, Bloom’s syndrome, and Fanconi anemia, as well as in some of the aforementioned chronic inflammatory diseases with autoimmune reactions.‘* Clastogenic factors exert genotoxic effects via the intermediacy of superoxide anion radicals. They induce not only chromosome mutations, but also gene mutation, as shown for the HPRT locus. Extracellular superoxide production is known to activate proto-oncogenes and to in-

Clastogenic

factors

duce mutations at the tumor suppressor loci via strand breakage and DNA sequence changes.27,28 Recent findings suggest involvement of reactive oxygen species in the activation of the pleiotropic transcriptional activator NF-kI3, resulting in expression of HIV-1.29.3* Since clastogenic factors are stimulating superoxide anion production by competent cells, they might also play a role in this activation process. High values for clastogenic plasma activity were observed in patients group A, indicating that the prooxidant state is an early event in HIV infection. Further work is in progress in order to define time correlations between appearance of clastogenic factors in the plasma and positivity for HIV infection. An example of interaction between clastogenic factors, superoxide anion production, and release of endogenous retroviruses is given by the animal model for autoimmune disease, the New Zealand Black mouse. This animal model for autoimmune disease harbors a xenotropic endogenous type C-virus” and exhibits increased chromosomal breakage, correlated with the presence of clastogenic factors in the bloodstream. By selective matings, according to chromosome breakage frequencies, two NZB sublines could be developed, which differ in clastogenic factor production, superoxide anion generation by peritoneal macrophages, xenotropic virus expression (demonstrated by reverse transcriptase levels), and the incidence of lymphomas and autoimmune hemolytic anemia at the age of 18 months.‘2-‘4 The chromosomal instability of NZB mice could be reduced to nearnormal values by intraperitoneal injection of SOD.35 Although HIV patients have significantly decreased plasma glutathione, total plasma thiols, ascorbate, glutathione peroxidase, and selenium, 6 plasma antioxidams did not differ significantly between low- and high-breakage groups, indicating that the clastogenic activity might not correlate with the antioxidant status. However, similar to the observations in the NZB mouse model and other conditions accompanied by clastogenic factors, SOD also inhibited the clastogenic effects of plasma ultrafiltrates from the HIV patients of this study. Other antioxidants have not yet been studied as anticlastogens, but their therapeutic or prophylactic efficacy might be evaluated using clastogenic factors as a guide as well in vitro as in vivo studies. Further studies are necessary to analyze the correlation between plasma antioxidants and clastogenic activity and to evaluate the clinical significance of increased clastogenic factors in HIV infection. REFERENCES 1. Sonnerborg, A.; Carlin, G.; Akerlund, B.; Jarstrand, C. Increased production of malondialdehyde in patients with HIV infection. J. Infect Dis. 20:287-290; 1988.

in HIV-

I patients

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29. Schreck, R.; Rieber, P.; Baeuerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-l. EMBO J. 10:2247-2258; 1991. 30. Schreck, R.; Albermann, K.; Baeuerle, P. A. Nuclear transcription factor kB: An oxidative stress responsive transcription factor of eukaryotic cells (a review). Free Rad. Res. Commun. 17:221-237; 1992. 31. Datta, K. S.; Schwartz, R. S. Genetics of expression of xenotropic virus and autoimmunity in NZB mice. Nature 263:412414; 1976. 32. Emerit, I.; Feingold, J.; Levy, A.; Martin, E.; Housset, E. Tumor incidence and development of autoimmune hemolytic anemia in two breeding lines of the NZB mouse strain, that differ in chromosome breakage. J. Natl. Cancer. Inst. 64:5 13 -5 18; 1980. 33. Bernard, C.; Emerit, I. Diverse xenotropic virus expression in two NZB mouse breeding lines that differ in chromosome breakage. J. Na?. Cancer Inst. 68:513-517; 1980. 34. Khan, S. H.; Emerit, I.; Feingold, J. Superoxide and hydrogen peroxide production by macrophages of NZB mice. Free Radic. Biol. Med. 8:339-345; 1990. 35. Emerit, I.; Levy, A.; Michelson, A. M. Effect of superoxide dismutase on the chromosomal instability of NZB mice. Cyrogenet. Cell Genet. 30:65-69; 1981.