Mutation Research, 283 (1992) 91-95
91
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0707
Inorganic arsenic effects on human lymphocyte stimulation and proliferation M . E . G o n s e b a t t a, L. V e g a
a
L . A . H e r r e r a a R. M o n t e r o a, E. R o j a s a n d P. O s t r o s k y - W e g m a n a
a
M . E . Cebrifin b
a Instituto de Investigaciones Biom~dicas, UNAM and h CINVESTAV,, IPN, Mexico D.F., Mexico
(Received 24 March 1992) (Revision received 25 May 1992) (Accepted 28 May 1992)
Keywords: Arsenic; Human lymphocyte proliferation; Cell cycle kinetics
Summary Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10 -7, 10 -8 and 10 -9 M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10 -9 M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and proliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.
Individuals exposed to high concentration of arsenic in drinking water develop skin lesions and skin cancer (Tseng, 1977; Cebri~n et al., 1983), similar to those reported by Walder et al. (1971) in patients taking immunosuppressive drugs. Furthermore, prolonged immunodeficiency increases the risk for selected tumor types (Zbinden, 1989;
Correspondence: Maria E. Gonsebatt, Instituto de Investigaclones Biom~dicas, UNAM, A.P. 70228, Mexico 04510, D.F., Mexico.
Sneller and Swober, 1990). Recently, we observed that lymphocyte cultures from individuals exposed to high levels of arsenic showed a slower cell cycle kinetics than cultures from low-exposed individuals (Ostrosky-Wegman et al., 1991). Since inhibition of lymphocyte proliferation has been used to identify agents and factors which depress the cellular immune response (McCarthy et al., 1990; Rodl et al., 1990; Orson et al., 1989; Moller et al., 1989), we decided to investigate the effects of pentavalent (As V) and trivalent (As III) arsenic on human lymphocyte stimulation and pro-
92
liferation, using arsenic concentrations similar to those found in the blood of highly exposed individuals (Vahter, 1988). Materials and methods
Two types of experiments were conducted. One experimental protocol was designed to study the effects of As III and As V on actively proliferating lymphocytes, the other was designed to evaluate the effects on lymphocytes about to initiate proliferation (i.e., while in the G 0- G ~ cell cycle phases). Sodium arsenate ( N a 2 H A s O a . 7 H 2 0 ) and sodium arsenite (NaAsO2),-both obtained from Sigma, were used as the source of As V and As III, respectively. The salts were dissolved in water and the solutions sterilized by filtration just before use.
Effects on proliferating lymphocytes Peripheral blood was obtained from healthy donors (two males aged 25 and 28 and two females aged 25 and 28). All donors were nonsmokers and had not taken any drugs for at least 1 week before sampling. Duplicate 72-h lymphocyte cultures were started as described: 0.5 ml of blood in 6 ml of RPMI culture medium supplemented with 0.01 ml/ml of L-glutamine and nonessential amino acids (Gibco), 32 /xM of bromodeoxyuridine (Sigma) and 0.2 ml of phytohemag-
glutinin (PHA) (Microlab). Cultures were incubated for 72 h at 37°C with exposure to arsenicals for the last 24 h before harvesting. The doses tested were 10 7, 10-8 and 10 -9 molar concentrations which are equivalent to blood total arsenic concentrations ranging from 13 to 0.13/xg/l for As III and from 31 to 0.31 ~g/1 for As V.
Effects on non-proliferating lymphocytes Whole blood lymphocyte cultures from two healthy donors (one male and one female) were exposed to trivalent and pentavalent arsenic at 10 -9 M for 2, 6 and 24 h from the time of the addition of PHA. Another set of cultures were exposed for 2, 6 and 24 h to As in the absence of PHA. At the end of the As exposure period, the control and treated cells were washed twice with RPMI 1640. PHA was added to all cultures, and the total incubation period in the presence of PHA was 72 h.
Harvesting and scoring Harvesting, coding of slide preparations and staining were performed as described elsewhere (Ostrosky-Wegman et al., 1986). The mitotic index (MI) was scored as the number of mitoses in 2000 mononuclear cells. For the cell cycle kinetics determination, the proportion of first (MI), second (M e) and third or more (M 3) cell division metaphases in 100 mitoses from each duplicate
TABLE 1 EFFECTS O F D I F F E R E N T C O N C E N T R A T I O N S O F A R S E N I C ON T H E A V E R A G E ( + SE) P R O P O R T I O N S OF Mr, M z AND M 3 A N D MI IN P R O L I F E R A T I N G CELLS Control
10 -9 M *
10 8 M *
10 -7 M *
As III M1 Me M3 %MI
13.2 40.9 45.9 2.49
+1.40 +1.05 -+2.00 _+0.58
23.7 _+3.00 40.1 _+2.25 36.2 +_2.45 2.74 _+0.50
34.7 -+4.15 41.2 -t-2.00 24.2 +2.35 1.91 +_0.48
52.7 _+7.2 31.8 -+3.2 15.5 -+3.6 1.40 -+0.27
As V Mi M2 M3 %MI
12.65 -+0.90 40.90 -+ 1.30 45.95 -+ 1.75 2.96-+0.16
20.25 _+0.75 42.75 -+ 0.37 37.00 -+0.50 2.60-+0.31
33.88 -+ 1.06 38.63 + 1.44 27.50 -+ 1.62 2.21 _+0.37
45.25 + 0.25 38.50 _+ 1.12 19.25 -+ 1.00 1.13+_0.16
* p < 0.05. %MI, mitotic index (percentage).
93
culture were determined. The replication index (RI) was calculated according to Ivett and Tice (1986).
%ofMI
RI
3,5"
2.5
3 2 25 1.5
2
Statistical analysis of results The average proportions of M 1, M 2 and M 3 were c o m p a r e d by a chi-square test while RI and MI were analyzed using a t-test.
1.5
1
1 05 05
10-9 M
Results
0 10-7 M
10-8 M
Molar ooncentrations --~-MIAsIII
The modifications in the proportions of M1, M 2 and M 3 in the cultures treated with different concentrations of As III and V were significant
~
MIA8V
-~RIASIII
*
RIAs
Fig. 1. Average replication (RI) and mitotic (MI) indexes in proliferating lymphocytes treated with different concentrations of sodium arsenite or sodium arsenate.
TABLE 2 E F F E C T O F 10 -9 M As T R E A T M E N T ON T H E A V E R A G E ( + SE) P R O P O R T I O N S O F M I, M e A N D M 3 A N D MI Length of exposure 2h
6h
24h
Control As III As V
13.00_+ 0.99 24.75 _+ 1.24 * 24.50 _+0 *
12.50_+ 1.49 38.75 _+9.24 * 33.25 _+2.74 *
18.00 -+ 0.99 73.00_+ 0.00 50.00 _+35.46
M2
Control As III As V
31.50 _+2.49 * 38.25 _+5.74 * 39.50_+0 *
39.50 _+4.49 34.50_+ 3.99 * 45.00_+3.99 *
37.00 _+ 4.99 9.00_+ 0.00 16.67_+20.47
M3
Control As III As V
55.50 _+ 1.49 37.00_+ 4.49 * 36.00 -+ 0 *
48.00 + 5.99 26.75 _+5.24 * 21.75 _+6.74 *
45.00 _+ 3.99 18.00-+ 0.00 33.34 + 40.94
%MI
Control As III As V
2.47 _+0.57 1.35 _+0.55 * 1.55 _+0.08 *
1.80 -+ 0.22 1.05 _+0.95 * 0.87 _+0.07 *
1.15 _+ 0.45 0 0
Ml
Control As III As V
11.50 _+3.50 22.00 _+2.49 * 19.75_+4.24 *
14.50 _+0.49 35.00 _+0.49 * 33.50_+2.99 *
16.00+ 0.99 44.00± 0 * 46.50+ 5.49
M2
Control As III As V
29.50 _+3.49 34.50+ 1.49 * 37.25 -+ 5.74 *
36.50 _+6.49 30.50-+0.99 * 37.75 -+ 2.74 *
36.00_+ 0.99 36.00 + 0 * 34.5 _+ 2.49
M3
Control As III As V
59.00 _+0 43.50_+0.99 * 43.00__+ 1.49 *
49.00 -+ 6.99 34.50_+ 1.49 * 28.75_+6.99 *
48.00+ 1.99 20.00_+ 0 * 19.00_+ 2.90
%MI
Control As III As V
2.38 _+0.20 1.48_+0.50 * 1.28_+0.45 *
1.82 _+0.12 1.51_+0.39 * 1.67_+0.26 *
2.97_+ 0.17 0.27_+ 0.22 0.67_+ 0.17
Without PHA M1
With PHA
* p < 0.05.
94 Of M1 80 _//J
70
~+
60 50 40 30 2C 10 i
0
I
I
!
J
I
i
I
~ ~
6
i
I 24
He of treatment --
Conlrol
~
AS III w o , P H A
A8 V w, ,3 PHA
~
AS V ~, PHA
~
AS III w P H &
Fig. 2. Percentage of first division m e t a p h s e s (M 1) in 72-h lymphocyte cultures treated during G 0 or. G 1 with 10 - 9 M sodium arsenite or 10 9 M sodium arsenate.
( p < 0.05) and dose related, as shown in Table 1. The decrease in the RI of proliferating lymphocytes was observed after treatment with arsenic levels above 10 - 9 M . A s III ( 1 0 - 7 M ) reduced the RI to 69% of control values (Fig. 1). The average MI obtained can also be seen in Fig. 1. The greatest delay in cell cycle kinetics corresponds to the lowest MI values which occurred at 10 - 7 M for both arsenic salts. No differences were observed between the degree of inhibition induced by the two As species. The time course of arsenic effects on the average proportions of M~, M 2 and M 3 and the MI, when lymphocytes were treated at the beginning of the cultures for 2, 6 and 24 h, is shown in Table 2. When MI was 0 (see Table 2), several slides had to be analyzed, and only 20-80 metaphases per culture could be scored. Cell proliferation was significantly affected following an exposure as short as 2 h to 10 - 9 M of As III and As V ( p < 0.05). The inhibition of stimulation and proliferation was greatest when lymphocytes were exposed to the element for 24 h at the onset of culture. Fig. 2 shows increased proportions of M 1 as result of longer treatments; a very small n u m b e r of mitoses per culture was found in the 24-h exposure in the absence of PHA. Discussion
Exposure of human lymphocytes in vitro to concentrations of inorganic arsenic similar to those found in the blood of exposed subjects
(Vahter, 1988) results in a depressed response to P H A stimulation and in a delayed cell cycle progression in a dose-related manner. The most sensitive stage for arsenic inhibition of stimulation and proliferation is during G o (Table 2 and Fig. 2), the cell cycle phase in which most peripheral blood lymphocytes circulate. Arsenite (As IlI) has been shown to bind to the SH groups of proteins (Vahter, 1988), which can result in the inhibition of thiol-containing enzymes such as the D N A ligases (Li and Rossman, 1989). Since very few lymphocytes were able to respond to P H A stimulation after a 24-h exposure to 10 -9 M arsenic, it is possible that other thiol-containing proteins besides ligases I and II are inactivated by interaction with arsenic. This effect was less pronounced in proliferating cells (Table 1), probably due to the fact that they were already stimulated and therefore in another cell cycle stage. Also, their more active protein synthesis and turnover could diminish the inhibitory effects of arsenic. A more potent toxicity of arsenite (As III) than arsenate (As V) has been reported (Jacobson-Kram and Montaibano, 1985; Vahter, 1988). A possible explanation for the similar toxicity of As III and As V found here could be that As V may have been reduced in the cultures to As III, since Bertolero et al. (1987) have observed this reduction to occur in an embryo cell line. Inorganic arsenic represented 20% of the total arsenic excreted in a group of individuals with 390 /.Lg As/1 in their drinking water (OstroskyWegman et al., 1991). According to Vahter (1988) and Valentine (1979), this kind of exposure elevates blood levels to 50-60 p~g As/1. The impairment of G 0 - G 1 lymphocytes to respond to P H A stimulation after in vitro exposure to concentrations as low as 0 . 1 3 / ~ g / l could provide an explanation for depressed growth characteristics observed for lymphocytes cultured from exposed subjects (Ostrosky-Wegman et al., 1991). This diminished T-lymphocyte response to P H A stimulation has also been observed in lymphocyte cultures from immunodepressed subjects (Moller et al., 1989; Orson et al., 1989) who are prone to develop certain malignancies, such as skin cancer (Penn, 1988). Therefore, the impaired immune response due to arsenic exposure could
95 p l a y a r o l e in t h e i n c r e a s e d i n c i d e n c e o f c a n c e r observed in the arsenic-exposed groups studied.
Acknowledgement We thank Dr. R. Tice for valuable discussions.
References Bertolero, F., G. Pozzi, E. Sabbioni and U. Saffiotti (1987) Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation, Carcinogenesis, 8, 803-808. Cebrifin, M.E., A. Albores, M. Aguilar and E. Blakely (1983) Chronic arsenic poisoning in the north of Mexico, Hum. Toxicol., 2, 121-133. Ivett, J.L., and R.R. Tice (1982) Average generation time: a new method of analysis and quantitation of cellular proliferation kinetics, Environ. Mutagen., 4, 358 (Abstract). Jacobson-Kram, D., and D. Montalbano (1985) The reproductive effects assessment group's report on the mutagenicity of inorganic arsenic, Environ. Mutagen., 7, 787-804. Li, J.-H., and T.G. Rossman (1989) Inhibition of DNA ligase activity by arsenite: a possible mechanism of its comutagenesis, Mol. Toxicol., 2, 1-9. McCarthy, M.A., J.P. Michalski, E.S. Sears and C.C. McCombs (1990) Inhibition of polyamine synthesis suppresses human lymphocyte proliferation without decreasing cytokine production or interleukine 2 receptor expression, Immunopharmacology, 20, 11-20. Moiler, J., B. Hoffman, E. Langhoff, K. Damgard Jacobsen, N. Odum, E. Dickmeiss, L.P. Ryder, O. Thastrup, O. Sharff and B. Foder (1989) Immunodeficiency after allogeneic bone marrow transplantation in man. Effect of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187) in vitro, Scand. J. Immunol., 30, 441447. Orson, F.M., C.K. Saadeh, D.E. Lewis and D.L. Nelson (1989) Interleukin 2 receptor expression by T cells in human aging, Cell. Immunol., 124, 278-291.
Ostrosky-Wegman, P., G. Garcfa, R. Montero, B. Pdrez Romero, R. Alvarez Chac6n and C. Cortinas de Nava (1986) Susceptibility to genotoxic effects of niclosamide in human peripheral lymphocytes exposed in vitro and in vivo, Mutation Res., 173, 81-87. Ostrosky-Wegman, P., M.E. Gonsebatt, R. Montero, L. Vega, H. Barba, J. Espinosa, A. Palao, C. Cortinas, G. GarcfaVargas, L.M. del Razo and M. Cebrifin (1990) Lymphocyte proliferation kinetics and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico, Mutation Res., 250, 477-482. Penn, I. (1988) Tumors of the immunocompromised patient, Annu. Rev. Med., 39, 63-73. Rodl, S., G. Fuchs, G. Khoshsorur, F. Iberer and K.H. Tscheliessnigg (1990) Lipoprotein-induced modulation of cyclosporine-A-mediated immunosuppression, Eur. J. Clin. Invest., 20, 248-252. Sneller, M.C., and W. Swober (1990) Abnormalities of lymphokine gene expression in patients with common variable immunodeficiency, J. Immunol., 144, 3762-3769. Tseng, W.-P. (1977) Effects and dose response relationships of skin cancer and blackfoot disease with arsenic, Environ. Health Perspect., 19, 109-119. Vahter, M.E. (1988) Arsenic, in: T.W. Clarkson, L. Friberg, G.F. Nordberg and P.R. Sager (Eds.), Biological Monitoring of Toxic Metals, Plenum, New York, pp. 303-321. Valentine, J.L., H.K. Kang and G. Spivey (1979) Arsenic levels in human blood, urine and hair in response to exposure via drinking water, Environ. Res., 20, 24-32. Walder, B.K., M.R. Robertson and J. Jeremy (1971) Skin cancer and immunosuppression, Lancet, ii, 1282-1203. Zbinden, G. (1990) The relationship between clinical immunology and classical experimental immunotoxicology, in: G.N. Volans, J. Sims, F.M. Sullivan and P. Turner (Eds.), Basic Science in Toxicology, Proceedings of the V International Congress of Toxicology. Taylor and Francis, London, pp. 344-353.
Communicated by J.M. Gentile
91
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0707
Inorganic arsenic effects on human lymphocyte stimulation and proliferation M . E . G o n s e b a t t a, L. V e g a
a
L . A . H e r r e r a a R. M o n t e r o a, E. R o j a s a n d P. O s t r o s k y - W e g m a n a
a
M . E . Cebrifin b
a Instituto de Investigaciones Biom~dicas, UNAM and h CINVESTAV,, IPN, Mexico D.F., Mexico
(Received 24 March 1992) (Revision received 25 May 1992) (Accepted 28 May 1992)
Keywords: Arsenic; Human lymphocyte proliferation; Cell cycle kinetics
Summary Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10 -7, 10 -8 and 10 -9 M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10 -9 M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and proliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.
Individuals exposed to high concentration of arsenic in drinking water develop skin lesions and skin cancer (Tseng, 1977; Cebri~n et al., 1983), similar to those reported by Walder et al. (1971) in patients taking immunosuppressive drugs. Furthermore, prolonged immunodeficiency increases the risk for selected tumor types (Zbinden, 1989;
Correspondence: Maria E. Gonsebatt, Instituto de Investigaclones Biom~dicas, UNAM, A.P. 70228, Mexico 04510, D.F., Mexico.
Sneller and Swober, 1990). Recently, we observed that lymphocyte cultures from individuals exposed to high levels of arsenic showed a slower cell cycle kinetics than cultures from low-exposed individuals (Ostrosky-Wegman et al., 1991). Since inhibition of lymphocyte proliferation has been used to identify agents and factors which depress the cellular immune response (McCarthy et al., 1990; Rodl et al., 1990; Orson et al., 1989; Moller et al., 1989), we decided to investigate the effects of pentavalent (As V) and trivalent (As III) arsenic on human lymphocyte stimulation and pro-
92
liferation, using arsenic concentrations similar to those found in the blood of highly exposed individuals (Vahter, 1988). Materials and methods
Two types of experiments were conducted. One experimental protocol was designed to study the effects of As III and As V on actively proliferating lymphocytes, the other was designed to evaluate the effects on lymphocytes about to initiate proliferation (i.e., while in the G 0- G ~ cell cycle phases). Sodium arsenate ( N a 2 H A s O a . 7 H 2 0 ) and sodium arsenite (NaAsO2),-both obtained from Sigma, were used as the source of As V and As III, respectively. The salts were dissolved in water and the solutions sterilized by filtration just before use.
Effects on proliferating lymphocytes Peripheral blood was obtained from healthy donors (two males aged 25 and 28 and two females aged 25 and 28). All donors were nonsmokers and had not taken any drugs for at least 1 week before sampling. Duplicate 72-h lymphocyte cultures were started as described: 0.5 ml of blood in 6 ml of RPMI culture medium supplemented with 0.01 ml/ml of L-glutamine and nonessential amino acids (Gibco), 32 /xM of bromodeoxyuridine (Sigma) and 0.2 ml of phytohemag-
glutinin (PHA) (Microlab). Cultures were incubated for 72 h at 37°C with exposure to arsenicals for the last 24 h before harvesting. The doses tested were 10 7, 10-8 and 10 -9 molar concentrations which are equivalent to blood total arsenic concentrations ranging from 13 to 0.13/xg/l for As III and from 31 to 0.31 ~g/1 for As V.
Effects on non-proliferating lymphocytes Whole blood lymphocyte cultures from two healthy donors (one male and one female) were exposed to trivalent and pentavalent arsenic at 10 -9 M for 2, 6 and 24 h from the time of the addition of PHA. Another set of cultures were exposed for 2, 6 and 24 h to As in the absence of PHA. At the end of the As exposure period, the control and treated cells were washed twice with RPMI 1640. PHA was added to all cultures, and the total incubation period in the presence of PHA was 72 h.
Harvesting and scoring Harvesting, coding of slide preparations and staining were performed as described elsewhere (Ostrosky-Wegman et al., 1986). The mitotic index (MI) was scored as the number of mitoses in 2000 mononuclear cells. For the cell cycle kinetics determination, the proportion of first (MI), second (M e) and third or more (M 3) cell division metaphases in 100 mitoses from each duplicate
TABLE 1 EFFECTS O F D I F F E R E N T C O N C E N T R A T I O N S O F A R S E N I C ON T H E A V E R A G E ( + SE) P R O P O R T I O N S OF Mr, M z AND M 3 A N D MI IN P R O L I F E R A T I N G CELLS Control
10 -9 M *
10 8 M *
10 -7 M *
As III M1 Me M3 %MI
13.2 40.9 45.9 2.49
+1.40 +1.05 -+2.00 _+0.58
23.7 _+3.00 40.1 _+2.25 36.2 +_2.45 2.74 _+0.50
34.7 -+4.15 41.2 -t-2.00 24.2 +2.35 1.91 +_0.48
52.7 _+7.2 31.8 -+3.2 15.5 -+3.6 1.40 -+0.27
As V Mi M2 M3 %MI
12.65 -+0.90 40.90 -+ 1.30 45.95 -+ 1.75 2.96-+0.16
20.25 _+0.75 42.75 -+ 0.37 37.00 -+0.50 2.60-+0.31
33.88 -+ 1.06 38.63 + 1.44 27.50 -+ 1.62 2.21 _+0.37
45.25 + 0.25 38.50 _+ 1.12 19.25 -+ 1.00 1.13+_0.16
* p < 0.05. %MI, mitotic index (percentage).
93
culture were determined. The replication index (RI) was calculated according to Ivett and Tice (1986).
%ofMI
RI
3,5"
2.5
3 2 25 1.5
2
Statistical analysis of results The average proportions of M 1, M 2 and M 3 were c o m p a r e d by a chi-square test while RI and MI were analyzed using a t-test.
1.5
1
1 05 05
10-9 M
Results
0 10-7 M
10-8 M
Molar ooncentrations --~-MIAsIII
The modifications in the proportions of M1, M 2 and M 3 in the cultures treated with different concentrations of As III and V were significant
~
MIA8V
-~RIASIII
*
RIAs
Fig. 1. Average replication (RI) and mitotic (MI) indexes in proliferating lymphocytes treated with different concentrations of sodium arsenite or sodium arsenate.
TABLE 2 E F F E C T O F 10 -9 M As T R E A T M E N T ON T H E A V E R A G E ( + SE) P R O P O R T I O N S O F M I, M e A N D M 3 A N D MI Length of exposure 2h
6h
24h
Control As III As V
13.00_+ 0.99 24.75 _+ 1.24 * 24.50 _+0 *
12.50_+ 1.49 38.75 _+9.24 * 33.25 _+2.74 *
18.00 -+ 0.99 73.00_+ 0.00 50.00 _+35.46
M2
Control As III As V
31.50 _+2.49 * 38.25 _+5.74 * 39.50_+0 *
39.50 _+4.49 34.50_+ 3.99 * 45.00_+3.99 *
37.00 _+ 4.99 9.00_+ 0.00 16.67_+20.47
M3
Control As III As V
55.50 _+ 1.49 37.00_+ 4.49 * 36.00 -+ 0 *
48.00 + 5.99 26.75 _+5.24 * 21.75 _+6.74 *
45.00 _+ 3.99 18.00-+ 0.00 33.34 + 40.94
%MI
Control As III As V
2.47 _+0.57 1.35 _+0.55 * 1.55 _+0.08 *
1.80 -+ 0.22 1.05 _+0.95 * 0.87 _+0.07 *
1.15 _+ 0.45 0 0
Ml
Control As III As V
11.50 _+3.50 22.00 _+2.49 * 19.75_+4.24 *
14.50 _+0.49 35.00 _+0.49 * 33.50_+2.99 *
16.00+ 0.99 44.00± 0 * 46.50+ 5.49
M2
Control As III As V
29.50 _+3.49 34.50+ 1.49 * 37.25 -+ 5.74 *
36.50 _+6.49 30.50-+0.99 * 37.75 -+ 2.74 *
36.00_+ 0.99 36.00 + 0 * 34.5 _+ 2.49
M3
Control As III As V
59.00 _+0 43.50_+0.99 * 43.00__+ 1.49 *
49.00 -+ 6.99 34.50_+ 1.49 * 28.75_+6.99 *
48.00+ 1.99 20.00_+ 0 * 19.00_+ 2.90
%MI
Control As III As V
2.38 _+0.20 1.48_+0.50 * 1.28_+0.45 *
1.82 _+0.12 1.51_+0.39 * 1.67_+0.26 *
2.97_+ 0.17 0.27_+ 0.22 0.67_+ 0.17
Without PHA M1
With PHA
* p < 0.05.
94 Of M1 80 _//J
70
~+
60 50 40 30 2C 10 i
0
I
I
!
J
I
i
I
~ ~
6
i
I 24
He of treatment --
Conlrol
~
AS III w o , P H A
A8 V w, ,3 PHA
~
AS V ~, PHA
~
AS III w P H &
Fig. 2. Percentage of first division m e t a p h s e s (M 1) in 72-h lymphocyte cultures treated during G 0 or. G 1 with 10 - 9 M sodium arsenite or 10 9 M sodium arsenate.
( p < 0.05) and dose related, as shown in Table 1. The decrease in the RI of proliferating lymphocytes was observed after treatment with arsenic levels above 10 - 9 M . A s III ( 1 0 - 7 M ) reduced the RI to 69% of control values (Fig. 1). The average MI obtained can also be seen in Fig. 1. The greatest delay in cell cycle kinetics corresponds to the lowest MI values which occurred at 10 - 7 M for both arsenic salts. No differences were observed between the degree of inhibition induced by the two As species. The time course of arsenic effects on the average proportions of M~, M 2 and M 3 and the MI, when lymphocytes were treated at the beginning of the cultures for 2, 6 and 24 h, is shown in Table 2. When MI was 0 (see Table 2), several slides had to be analyzed, and only 20-80 metaphases per culture could be scored. Cell proliferation was significantly affected following an exposure as short as 2 h to 10 - 9 M of As III and As V ( p < 0.05). The inhibition of stimulation and proliferation was greatest when lymphocytes were exposed to the element for 24 h at the onset of culture. Fig. 2 shows increased proportions of M 1 as result of longer treatments; a very small n u m b e r of mitoses per culture was found in the 24-h exposure in the absence of PHA. Discussion
Exposure of human lymphocytes in vitro to concentrations of inorganic arsenic similar to those found in the blood of exposed subjects
(Vahter, 1988) results in a depressed response to P H A stimulation and in a delayed cell cycle progression in a dose-related manner. The most sensitive stage for arsenic inhibition of stimulation and proliferation is during G o (Table 2 and Fig. 2), the cell cycle phase in which most peripheral blood lymphocytes circulate. Arsenite (As IlI) has been shown to bind to the SH groups of proteins (Vahter, 1988), which can result in the inhibition of thiol-containing enzymes such as the D N A ligases (Li and Rossman, 1989). Since very few lymphocytes were able to respond to P H A stimulation after a 24-h exposure to 10 -9 M arsenic, it is possible that other thiol-containing proteins besides ligases I and II are inactivated by interaction with arsenic. This effect was less pronounced in proliferating cells (Table 1), probably due to the fact that they were already stimulated and therefore in another cell cycle stage. Also, their more active protein synthesis and turnover could diminish the inhibitory effects of arsenic. A more potent toxicity of arsenite (As III) than arsenate (As V) has been reported (Jacobson-Kram and Montaibano, 1985; Vahter, 1988). A possible explanation for the similar toxicity of As III and As V found here could be that As V may have been reduced in the cultures to As III, since Bertolero et al. (1987) have observed this reduction to occur in an embryo cell line. Inorganic arsenic represented 20% of the total arsenic excreted in a group of individuals with 390 /.Lg As/1 in their drinking water (OstroskyWegman et al., 1991). According to Vahter (1988) and Valentine (1979), this kind of exposure elevates blood levels to 50-60 p~g As/1. The impairment of G 0 - G 1 lymphocytes to respond to P H A stimulation after in vitro exposure to concentrations as low as 0 . 1 3 / ~ g / l could provide an explanation for depressed growth characteristics observed for lymphocytes cultured from exposed subjects (Ostrosky-Wegman et al., 1991). This diminished T-lymphocyte response to P H A stimulation has also been observed in lymphocyte cultures from immunodepressed subjects (Moller et al., 1989; Orson et al., 1989) who are prone to develop certain malignancies, such as skin cancer (Penn, 1988). Therefore, the impaired immune response due to arsenic exposure could
95 p l a y a r o l e in t h e i n c r e a s e d i n c i d e n c e o f c a n c e r observed in the arsenic-exposed groups studied.
Acknowledgement We thank Dr. R. Tice for valuable discussions.
References Bertolero, F., G. Pozzi, E. Sabbioni and U. Saffiotti (1987) Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation, Carcinogenesis, 8, 803-808. Cebrifin, M.E., A. Albores, M. Aguilar and E. Blakely (1983) Chronic arsenic poisoning in the north of Mexico, Hum. Toxicol., 2, 121-133. Ivett, J.L., and R.R. Tice (1982) Average generation time: a new method of analysis and quantitation of cellular proliferation kinetics, Environ. Mutagen., 4, 358 (Abstract). Jacobson-Kram, D., and D. Montalbano (1985) The reproductive effects assessment group's report on the mutagenicity of inorganic arsenic, Environ. Mutagen., 7, 787-804. Li, J.-H., and T.G. Rossman (1989) Inhibition of DNA ligase activity by arsenite: a possible mechanism of its comutagenesis, Mol. Toxicol., 2, 1-9. McCarthy, M.A., J.P. Michalski, E.S. Sears and C.C. McCombs (1990) Inhibition of polyamine synthesis suppresses human lymphocyte proliferation without decreasing cytokine production or interleukine 2 receptor expression, Immunopharmacology, 20, 11-20. Moiler, J., B. Hoffman, E. Langhoff, K. Damgard Jacobsen, N. Odum, E. Dickmeiss, L.P. Ryder, O. Thastrup, O. Sharff and B. Foder (1989) Immunodeficiency after allogeneic bone marrow transplantation in man. Effect of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187) in vitro, Scand. J. Immunol., 30, 441447. Orson, F.M., C.K. Saadeh, D.E. Lewis and D.L. Nelson (1989) Interleukin 2 receptor expression by T cells in human aging, Cell. Immunol., 124, 278-291.
Ostrosky-Wegman, P., G. Garcfa, R. Montero, B. Pdrez Romero, R. Alvarez Chac6n and C. Cortinas de Nava (1986) Susceptibility to genotoxic effects of niclosamide in human peripheral lymphocytes exposed in vitro and in vivo, Mutation Res., 173, 81-87. Ostrosky-Wegman, P., M.E. Gonsebatt, R. Montero, L. Vega, H. Barba, J. Espinosa, A. Palao, C. Cortinas, G. GarcfaVargas, L.M. del Razo and M. Cebrifin (1990) Lymphocyte proliferation kinetics and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico, Mutation Res., 250, 477-482. Penn, I. (1988) Tumors of the immunocompromised patient, Annu. Rev. Med., 39, 63-73. Rodl, S., G. Fuchs, G. Khoshsorur, F. Iberer and K.H. Tscheliessnigg (1990) Lipoprotein-induced modulation of cyclosporine-A-mediated immunosuppression, Eur. J. Clin. Invest., 20, 248-252. Sneller, M.C., and W. Swober (1990) Abnormalities of lymphokine gene expression in patients with common variable immunodeficiency, J. Immunol., 144, 3762-3769. Tseng, W.-P. (1977) Effects and dose response relationships of skin cancer and blackfoot disease with arsenic, Environ. Health Perspect., 19, 109-119. Vahter, M.E. (1988) Arsenic, in: T.W. Clarkson, L. Friberg, G.F. Nordberg and P.R. Sager (Eds.), Biological Monitoring of Toxic Metals, Plenum, New York, pp. 303-321. Valentine, J.L., H.K. Kang and G. Spivey (1979) Arsenic levels in human blood, urine and hair in response to exposure via drinking water, Environ. Res., 20, 24-32. Walder, B.K., M.R. Robertson and J. Jeremy (1971) Skin cancer and immunosuppression, Lancet, ii, 1282-1203. Zbinden, G. (1990) The relationship between clinical immunology and classical experimental immunotoxicology, in: G.N. Volans, J. Sims, F.M. Sullivan and P. Turner (Eds.), Basic Science in Toxicology, Proceedings of the V International Congress of Toxicology. Taylor and Francis, London, pp. 344-353.
Communicated by J.M. Gentile
