Immunopharmacology and immunotoxicity of zinc diethyldithiocarbamate

Int. J. lrnmunopharmac, Vol 10, No. 4, pp. 4 8 9 - 4 9 3 , 1988. Printed in Great Britain

0 1 9 2 - 0 5 6 1 / 8 8 $3 00 + .00 © 1988 lrtternatlonal Society for lrnmunopharmacology

SHORT COMMUNICATION I M M U N O P H A R M A C O L O G Y A N D I M M U N O T O X I C I T Y OF ZINC DIETHYLDITHIOCARBAMATE GERARD RENOUX, MICHELINE RENOUX and JEAN-MAURICEGUILLAUMIN Laboratoire d'Immunologie, Facult6 de M6dicine, 37000 Tours, France (Received 12 November 1987 and in final form 10 February 1988)

Zinc is essential for the functions of the immune system, and sodium diethyldithiocarbamate (imuthiolTM)restores and regulates the numbers and activities of cells of the T-cell lineage. The combination of both elements was therefore tested for immune enhancement and immunotoxicity. The data presented herein show that the administration of zinc diethyldithiocarbamate was devoid of immunoenhancing influence on the responses to T-cell mitogens, and exerted a cytocidal effect on spleen lymphocytes.

Abstract --

Zinc deficiency affects host immune resistance and a strict physiological requirement for zinc has been found to be essential for both primary and secondary antibody responses, numbers of cells bearing the Thy-1 phenotype, cell-mediated cytotoxicity, NK activity, B-cell development, and macrophage activities (see, Winchurch, 1987). Purified sodium diethyldithiocarbamate (imuthiol~M), on the other hand, evinces distinctive properties in recruitment, maturation and regulation on the T-cell lineage. Imuthiol is neither a mitogen, a polyclonal activator, nor an interferon inducer, It induces prothymocytes to mature into functionally active T-cells, and T-cells to generate enhanced levels of responses to mitogens and alloantigens, cytotoxic activities and IL-2 production. It increases NK activity. Through its effects on T-cells, B-cells are induced to secrete primary antibodies of the IgG class and monocyte/ macrophages to participate in delayed-type hypersensitivities, and to increased IL-I production (Bruley-Rosset, Vergnon & Renoux, 1986; Chung, Florentin & Renoux, 1985; Mossalayi, Descombe, Musset & Goube de Laforest, 1985; Pompidou, Duchet, Cooper, Mac6, Telvi, Coutance, Hadden & Renoux, 1985; Renoux, 1982, 1985, 1987; Renoux & Renoux, 1979, 1984). We thought, therefore, that it could be of interest to evaluate the influence of zinc diethyldithiocarbamate (Zn-DTC) on the responses to mitogenic lectins, since lymphocyte activation by these lectins displays the same accessory cell requirements as antigen489

induced triggering (Habu & Raff, 1977), and may thus provide a good model system for determination of an enhancing effect. In vitro cytotoxicity was evaluated on murine spleen cells co-cultured with various amounts of Zn-DTC; in vivo cytotoxicity was evaluated at 1 - 8 day time intervals after subcutaneous (s.c.) injections of various doses of Zn-DTC. The present findings show that, in contrast to sodium diethyldithiocarbamate, Zn-DTC is devoid of enhancing properties on the polyclonal responses to the T-cell mitogens, Con A and PHA, but rather exerts a cytocidal effect on spleen lymphocytes.

EXPERIMENTAL

PROCEDURES

Mice

Inbred female, BALB/c, C57B1/6 and nu/nu (BALB/c background) mice were obtained from the Centre de Selection des Animaux de Laboratoire (CSAL, CNRS, Orleans, France). They were maintained in an air-filtered and conditioned room at 24°C, and partially protected from environmental infections by Isocap filters (Iffa-Credo, L'Arbresle, France). N u / n u mice exhibiting abnormal features such as weight loss during quarantine, or revealing gross or microscopic liver lesions at autopsy were excluded from the experiments, as potentially infected (Scheid, Goldstein & Boyse, 1975).

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Treatments Zinc diethyldithiocarbamate (Zn-DTC), mol. wt 219, was kindly provided by C. C h a r b o n n i e r (Institut M6rieux, Lyon). It was suspended in sterile,pyrogenflee, saline, b u f f e r e d at p H 7.4, and injected s.c. The doses and time intervals are indicated in the Results section.

Cell counts Mice were sacrificed by cervical dislocation, under brief ether anesthesia. Spleens were removed aseptically, and cell suspensions were prepared by teasing in 1 ml o f cold Eagle's minimal essential m e d i u m and passing the separated cells through a stainless steel mesh. Total nucleated cell n u m b e r s were counted in a Model F counter (Coulter Electronics), and the percentages o f viable cells determined by t r y p a n blue exclusion.

RPMI-1640 containing 5% inactivated fetal calf serum (Gibco), 2 mM glutamine, 100 U / m l penicillin and 100/ag/ml streptomycin. Then, 0.2 ml aliquots o f the mixture, containing 0.5 × 106 cells, were a d d e d to wells o f sterile f l a t - b o t t o m microplates (Falcon Division B e c t o n - D i c k i n s o n , Oxnard, CA). Triplicate cultures were incubated for 48 h, at which time 0.5/aCi 3H-thymidine was a d d e d to each well. A f t e r an additional 18 h, the cultures were harvested o n t o glass fiber filters with the aid o f a M A S H II harvester, and 3H-thymidine incorporation was determined by liquid scintillation spectrometry.

Statistical evaluation S t u d e n t ' s t-test was used for determination o f the significance o f mean differences between groups.

Assay f o r D N A synthesis

RESULTS

Spleen cell suspensions were adjusted to 5 × 1 0 6 viable cells/ml and mixed with an equal volume o f medium containing either 2 tag/ml phytohemagglutinin ( P H A , Wellcome, Beckenham, England), 4 / a g / m l concanavalin A (Con A, Calbiochem) or 10/ag/ml p o k e w e e d m i t o g e n ( P W M , IBF, Clichy, France) in

Kinetic influences on body and organ weights, and on spleen cell vhTbility The influences o f Z n - D T C , in a range o f doses from 0.1 to 5.0 m g / k g , on body and spleen weights

Table 1. Kinetic influences of Zn-diethyldithiocarbamate on body and spleen weights, and on spleen cell viabihty Treatment (mg/kg) and time interval 0.1 Day 1 Day 4 Day 6 Day 8 1.0 Day 1 Day 4 Day 6 Day 8 2.5 Day l Day 4 Day 6 Day 8 5.0 Day 1 Day 4 Day 6 Day 8 Controls

Body weight (g)

Spleen weight (mg)

Spleen cells Total × 106

% live

21.3 20.8 19.5 19.7

___ 1.4 ~ 0.8* _+ 1.2" ~ 2.1

76 78 67 74

_+ 9 _+ 7 _+ 7 _+ 16

118.8 105.0 124.0 120.0

__. 9 ± 8* ± 8 .+ 3

67 61 74 72

_+ 3* _+ 3* _ 2* .+ 4*

21.3 20.9 18.8 20.1

.+ 1.4 ± 1.3 _+ 1.7" .+ 0.7*

76 78 64 82

.+ 9 __. 7 _+ 18 _+ 17

128.1 105.0 110.0 118.0

_+ 5 .+ 8* _+ 3 _+ 2

66 60 80 71

_+ 3* .+ 4* +__3* _+ 5*

22.2 19.8 21.3 22.4

_+ 0.4 .+ 0.1" _+ 0.4 _+ 1.6

95 66 76 98

_+ 17 -+ 23 + 15 -+ 13

126.0 .+ 5 99.2 _+ 10" 124.0 .+ 3 132.2 _ 9

68 78 71 67

_+ 4* -+ 5* .+ 2* _+ 2*

19.4 22.6 21.7 21.7 22.1

_+ 0.5* .+ 1.2 _+ 4.2 _+ 0.5 _ 0.8

83 86 78 76 76

_+ 9 -+- 18 .+ 13 .+ 15 + 8

124.0 +_ 7 139.4 _+ 10" 145.0 _+ 9* 125.3 +_ 10 116.8 _+ 6

71 68 61 66 92

_+ 3* _+ 7* _+ 7* _+ 2* .+ 5

Data expressed as mean values _+ S.E.M. of five mice individually tested in each experimental group. * Significantly different from controls (P ~< 0.01).

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Table 2. In vivo kinetic influences of Zn-diethyldithiocarbamate on mitogen-induced lymphoproliferation Mitogen added*

Treatment (mg/kg) a n d time interval

None

0.1 Day 1 Day 4 Day 6 Day 8 1.0 Day 1 Day 4 Day6 Day 8 2.5 Day 1 Day 4 Day 6 Day 8 5.0 Day 1 Day 4 Day 6 Day 8 Controls

10.1 14.1 5.3 3.5

PHA

Con A

± ± ± ±

PWM

± ± ± ±

1.4 t 1.3 t 0.4 * 0.8

184.7 181.1 163.0 160.6

± 10.7 ± 8.3 ± 8.4 ± 15.9

58.4 45.5 43.7 41.7

5.7 5.0 9.0 6.5

71.9 60.6 53.3 27.5

± ± ± ±

5.5* 5.6 t 2.6 t 4.0

13.5 ± 9.6 ± 5.8± 7.3 ±

2.0 t 1.5 ~ 1.2 t 1.0 *

174.9 145.5 158.1 189.7

± 17.7 ± 4.7* ± 8.1 ± 14.8

29.1 ± 2.0* 50.1 ± 4.6 55.8±4.3 58.3 ± 3.0

54.3 _+ 3.7 * 44.2 ± 3.7 t 6 0 . 6 ± 5.7 t 40.0 ± 4.6 t

6.5 5.5 4.2 8.1

± ± ± ±

1.3 t 0.7 t 1.0 2.5 t

113.6 137.9 132.5 117.6

± 10.1' ± 6.5* ± 8.6* ± 5.9*

33.6 31.4 33.9 25.4

± ± ± ±

4.3* 6.8* 2.8* 3.7*

31.7 31.6 50.2 34.8

± 2.9 ± 2.4 _+ 6.3 t ± 6.8

3.0 12.8 5.5 5.9 3.3

± ± ± ± ±

0.3 1.7* 0.4 t 1.4 e 0.5

123.7 168.5 168.6 158.0 162.4

± 6.3* ± 7.2 ± 11.2 ± 11.6 ± 8.0

38.7 58.8 57.7 23.4 50.5

_+ 3.0* ± 5.4 ± 4.8 ± 2.2* ± 4.0

27.3 52.7 55.1 28.9 27.7

± + ± ± ±

4.2 2.2* 3.1 t 5.4 2.2

* Mean c o u n t s / m i n ± S.E.M. of 15 observations in each experimental group. t Significantly increased above controls (P ~< 0.01); * significantly decreased below controls (P ~< 0.01).

Table 3. In vitro mitogenic influence o f Zn-diethyldithiocarbamate Mouse strain BALB/c C57B1/6

Nu/nu

0 6343± 628 16,165± 466 12,870± 2894

10 -~

10 4

Concentration (mg/ml)* 10-5 10 6

99± 26 157± 77 325± 105

392± 126 205± 112 129± 101

1384± 240 8595± 1997 1306± 71

6352± 431 17,801± 1650 6963± 2200

10-7 6903± 201 16,416± 2843 12,772± 1752

10 8 9186± 1769 15,172± 1030 13,813± 1164

10-9

8447± 152 16,370± 1471 12,222± 2894

Mean c o u n t s / m i n _+ S.E.M. o f triplicate assays of three spleen cell suspensions individually tested in each experimental group. * A m o u n t s added to 106 live splenocytes in 1 ml of supplemented R P M I medium.

a n d o n t o t a l s p l e e n cell c o u n t s a n d GT0 live cells w a s assessed on days 1 - 8 post-treatment. Table 1 refers to p o o l e d v a l u e s f o r five m i c e in e a c h e x p e r i m e n t a l g r o u p . It s h o w s t h a t t h e a g e n t h a s i n d u c e d v a r i a b l e , a l t h o u g h s m a l l , c h a n g e s in m e a n b o d y w e i g h t , b u t d i d n o t a f f e c t s p l e e n w e i g h t s . V a r i a b l e c h a n g e s in t h e t o t a l n u m b e r s o f n u c l e a t e d s p l e e n cells w e r e a l s o o b s e r v e d ( T a b l e 1). Importantly, the administration of Zn-DTC had a significant cytocidal influence on murine l y m p h o c y t e s (a m e a n 6 7 % live cells, as c o m p a r e d to 92O7o live cells in s a l i n e - t r e a t e d c o n t r o l s ; P < 0.01).

The cytotoxic effect was independent of the dose and l a s t e d f o r at l e a s t 8 d a y s .

Kinetics o f the responses to mitogens on Z n - D T C treated mice In this experiment, BALB/c mice were injected s.c. w i t h v a r i o u s d o s e s o f Z n - D T C (five m i c e p e r dose and day of sampling). After 1 day, 4 days, 6 d a y s a n d 8 d a y s , r e s p e c t i v e l y , 0.5 × 10 6 live ( t r y p a n b l u e a s s a y ) s p l e e n cells f r o m e a c h m o u s e w e r e individually assayed for their proliferative responses to PHA, Con A and PWM. The lectin

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concentrations employed were determined as suboptimal in preliminary assays. As shown in Table 2, Zn-DTC increased spontaneous 3H-thymidine incorporation in the absence of lectins and independent of the time interval. In contrast, ZnDTC did not increase above control values the proliferative responses to P H A and Con A. Even more, the 2.5 mg/kg dose significantly impaired the responses to these T-cell mitogens in comparison to saline-treated control mice (P < 0.001), during the whole observation period. However, PWM responses were clearly enhanced, particularly with the 1.0 mg/kg dose at all observation intervals (P < 0.01 to controls), for up to day 6 with the 0.1 dose and at day 6 and 8 with the 5.0 mg/kg dose, and none of the doses of Zn-DTC inhibited the responses to PWM.

In vitro mitogenic influence o f Z n - D T C Since zinc salts are inducers of non-specific stimulation of human lymphocytes (Kirchner & Ruhl, 1970), the in vitro proliferative influence of various concentrations of Zn-DTC was assayed on BALB/c, C57B1/6 and nu/nu (BALB/c background) spleen lymphocytes. The findings in 24 h cultures of drug-treated cells and controls are summarized in Table 3. The concentrations of ZnDTC, between 10 -3 and 10 -5 m g / m l (1 /~g to 10 ng/ ml) noticeably inhibited the spontaneous DNA incorporation of cells from euthymic mice below that of untreated cell suspensions (P < 0.001), and the inhibitory effect extended to one more dilution (1 ng/ml) on nude cells. These inhibitions correlated with important cell death, as only 2 0 - 35°70 of cells remained viable by the trypan blue assay, in comparison with >78% in controls. The concentrations from 1 ng to 1 pg were slightly, although significantly ( / ~ 0.05), mitogenic for BALB/c lymphocytes, but not for the other cell suspensions. Live cell percentages were, however, similar to that of untreated controls in cocultures with the lowest Zn-DTC concentrations.

DISCUSSION The aim of the present study was to determine whether Zn-DTC could be a valuable immunotherapeutic agent, by combining in a single compound the immunorestorative properties of zinc and sodium diethyldithiocarbamate. Unexpectedly, an inhibition of the responses to the T-cell mitogens, P H A and Con A, contrasting with increased spontaneous and PWM-induced DNA incorporations was observed in spleen lymphocytes from Zn-DTC treated mice. The findings suggest that T-cells might have been the main target for the cytocidal activity of Zn-DTC, as PWM is generally accepted as a B-cell mitogen for mouse cells and as zinc can act as a B-cell mitogen (CunninghamRundles, Cunningham-Rundles, Dupont & Good, 1980). The influence of zinc deficiency on immune responses is well documented (Fernandes, Nair, Onoe, Tanaka, Floyd, & Good, 1979; Winchurch, 1987). However, zinc supplementation in otherwise adequately fed animals depresses neutrophil and macrophage motility, phagocytosis and intracellular bacterial killing, fails to alter the responses in mixed lymphocyte cultures, and increases plaque-forming cell responses in old, but not in young, animals (Chandra & Teijpar, 1983; Koller, 1980; Winchurch, 1987). These findings indicate that more knowledge needs to be accumulated concerning zinc deficiency and excess before its effects on the immune system can be fully assessed. Zinc is a tightly bound metal on many enzymes (particularly DNA and RNA polymerases) and proteins, and is toxic for lymphocytes at greater than optimal stimulatory concentrations, which stimulant effects are limited to 5 - 10°70 of the total lymphocyte population (Loeb, 1974). It might be, therefore, that the addition or administration of Zn-DTC has locally created an excess of zinc concentration on the lymphocyte membranes, as a consequence of the lipophilic properties of dithiocarbamates (Thorn & Ludwig, i962), thus inducing cytotoxicity and impairment of DNA incorporation.

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