Bioscience Reports, Vol. 10, No. 4, 1990
Direct Oxidation of Lymphocytes by
Chromic Acid Does Not Induce Blastogenesis
George L Malinin, i Francis J. Hornicek 2 and
Theodore I. Malinin 2Received February 28, 1990
Blastogenic and cytotoxic effects of hexavalent chromium were evaluated by direct, 2 and 20 min
oxidation of lymphocytes by 10.0 to 0.0005 mM CrO 3 at 0~ Oxidized cells exhibited concentrationdependent cytotoxicity and the inhibition of tritiated thymidine incorporation rates. When lymphocytes were oxidized first by 1.0mM periodic acid (H5IO6) and thereafter by 1.0 mM CrO3, the
viability and [3H]-TdR incorporation rates of sequentially oxidized cells were identical to the
corresponding indicators of lymphocytes oxidized only by CrO 3. The reversal of the oxidation
sequence restored [3H]-TdR incorporation to control levels and increased cell survival. It is therefore
concluded that direct interaction of hexavalent CrO 3 with plasma membrane of lymphocytes results in
concentration-dependent cytotoxicity and the inhibition of [3H]-TdR incorporation, but it does not
induce blastogenesis.
KEY WORDS: lymphocytes; blastogenesis; chromic acid; periodic acid; membrane oxidation; sialic
acid.
INTRODUCTION
C h r o m i u m is k n o w n to interact with a large n u m b e r of biological targets in v i v o
and in vitro [1]. T h e extent and c o n s e q u e n c e s of c h r o m i u m interaction with
biological systems d e p e n d on n u m e r o u s factors such as c o n c e n t r a t i o n , d u r a t i o n of
exposure, the p H of the reaction m e d i a and m o s t importantly, the oxidation state
of the c h r o m i u m c o m p o u n d s in question [2].
A l t h o u g h extensive data are available concerning c h r o m i u m m e t a b o l i s m and
toxicity [1, 2], in m a n y instances the initial m e c h a n i s m and s u b s e q u e n t effects o f
c h r o m i u m interactions with biological systems r e m a i n obscure. F o r instance,
while hexavalent ( C r V I ) and trivalent ( C r I I I ) c h r o m i u m induce mitogenesis and
sister c h r o m a t i d e x c h a n g e in l y m p h o c y t e s [3-6], the m e c h a n i s m o f mitogenic
stimulation exerted by c h r o m i u m o n l y m p h o c y t e s was n o t elucidated.
Department of Physics, Georgetown University, Washington, DC 20057 and 2 Surgical Research
Laboratories, Department of Surgery, University of Miami School of Medicine, Miami FL 33101.
347
0144-8463/90/0800-0347506.00/0~) 1990 Plenum Publishing Corporation
348
Malinin, Hornicekand Malinin
It is well established that oxidation of lymphocytes by periodic acid--a
reagent for the oxidative cleavage of 1,2-diols [7J--results in the formation of
plasma membrane-bound carbonyl groups which trigger mitogenesis [8-11]. The
most likely sites of periodate-generated carbonyls are the C7-C8 and C8-C9
hydroxyls of N-acetyl-neuraminic acid [9], an integral component of plasma
membrane glycoproteins in lymphocytes [12].
Chromium(VI) oxide is a powerful oxidant of alchols, diols, aldehydes and
ketones [13], and therefore it should oxidize 1,2-diols of N-acetylneuraminic acid.
If the oxidation of lymphocytes by chromic acid under physiologic conditions
indeed generates membrane-bound carbonyls, then the blastogenic response of
the oxidized cells can be expected. Alternatively, if the oxidation of lymphocytes
fails to generate carbonyl groups, then the oxidized cells will remain quiescent.
These considerations prompted us to investigate whether direct oxidation of
lymphocytes by chromium(VI) oxide induces blastogenesis.
MATERIALS
Chromium trioxide (CrO3), trypan blue, and paraperiodic acid (H5IO6) were
purchased from Fisher Scientific Co., Silver Springs, MD. Grand Island Biological Co. (GIBCO) supplied RPMI-1640 powered medium, fetal calf serum (FCS;
heat-inactivated for 30min at 56~
penicillin and Dulbecco's Ca 2+ and
Mgi+-free phosphate buffered saline (PBS). Streptomycin sulfate and sodium
bicarbonate (NaHCO3) were bought from Eli Lilly and Co., Indianapolis, In, and
J. T. Baker Co., Phillipsburg, NJ, respectively. The lectin, Concanavalin A (Con
A) was purchased from Sigma Chemical Co., St. Louis, MO, while methyltritiated thymidine ([3H]-TdR; spec. act., 70 GBq/mmol, i.e. 1.9 Ci/mmol) came
from Schwartz Mann, Orangeburg, NY. All plasticware was supplied by either
Corning, Corning NY, or Falcon, Oxnard, CA, and 4 to 6 weeks old female
Balb/C mice were purchased from Charles River, MA. Seventy-four micron
nylon mesh was bought from Small Parts, Inc., Miami, FL.
METHODS
Cell Cultures
Mouse spleen cell suspensions were prepared by gentle teasing of the excised
spleens with a rubber policeman, washing the dispersed pulp with PBS and by
filtration through 74 #m nylon mesh. Lymphocytes were pooled by density
gradient centrifugation on Histopaque [14], while the erythrocytes were lysed for
10 min in 0.1 M NHaC1 at 4~
Thereafter, the nucleated cells were enumerated by a ZBI Coulter Counter
and their viability assessed by trypan blue exclusion. After the experimental
procedures listed below, 5 x 10 6 cells/ml were cultured in RPMI-1640 medium
containing 10% v/v FCS, 100U/ml penicillin, 100ug/ml streptomycin and
Chromic Acid and Blastogenesis
349
1.8; mg/ml NaHCO3 at 37~ in a 5% CO2 humidified atmosphere. Following a
48 hr culture period, lymphocytes were pulsed for 16 hrs with 1 ~ Ci of [3H]-TdR
in 20 #1 of medium and then harvested onto glass fiber sheets for the subsequent
determination of their radioactivity.
The stimulation index--SI = (cpm [3H]-TdR incorporated by experimental
cells-cpm [3H]-TdR incorporated by control cells) was calculated for a constant
number of viable cells at 72 hrs. Each experiment was performed in quadruplicate
and viability determinations were performed in duplicate.
Oxidation of Lymphocytes by H510 6 and their Stimulation with Con A
Oxidation of lymphocytes by H5IO6 was performed as described previously
[10, 11]. An equal volume of cold (4~ 2 mM HsIO 6 in Ca 2§ and Mg2+-free PBS
was added to 5 • 106 lymphocytes/ml suspended in 4~ PBS. After 20rain
oxidation in an ice bath, the reaction mixture was diluted ten fold with cold
RP'MI-1640 and the oxidized cells were immediately centrifuged for 10 min at
300 g. The cells were then resuspended in fresh 4~ PBS and centrifuged again as
before. Thereafter, washed cells were cultured as stated. Positive controls for
H51[O6 and CrO3-induced effects were provided by lymphocytes stimulated with
2.5 #g/ml of Con A under standard conditions [15].
Oxidation of Lymphocytes by Cr03
Stock 20mM chromic acid was prepared in Ca 2§ and Mg2+-free PBS,
buffered with 15mg/ml of NaHCO3. Working 10.0mM, 1.0mM, 0.1mM,
0.01mM, 0.005mM, 0.001mM and 0.0005mM solutions were prepared by
diluting the stock solution with the requisite amount of PBS. All solutions were
kept in a refrigerator in the dark for no longer than 7 days before use.
Oxidation of 5 • 106 lymphocytes/ml was performed at 0~ for 0, 2 and
20 rain. The oxidation time is defined as the termination of oxidation immediately
upon dilution of Cr(VI) solutions with excess medium. Oxidation reactions were
stopped by 10 fold dilution of the reaction mixture with RPMI-1640 medium and
immediately thereafter the cells were centrifuged for 10 min at 300 g. The cell
pellets were then resuspended in PBS and centrifugation repeated. The cells
washed twice with PBS were then cultured and processed further as stated.
Tandem Oxidation of Lymphocytes by HslO 6 and by Cr03
Suspensions of 2.5 • 106 lymphocytes/ml were initially oxidized for 20 min by
1.0 mM HsIO 6 as stated previously, washed twice with PBS and oxidized again by
1.0mM f r O 3 for 20min. Oxidized cells were resuspended in RPMI-1640
medium, centrifuged and then cultured as stated.
The reverse order of the oxidation reactions was also performed in exactly
the same manner. Thereafter, sequentially oxidized cells were cultured for 72 hr
in RPMI-1640 medium and pulsed with 1/~ Ci of [3H]-TdR in 20/~1 of medium
during the last 16 hr in culture.
350
Malinin, Hornicek and Malinin
RESULTS
The initial ( " 0 " hr) interaction of lymphocytes with the indicated CrO3
solutions (Table 1) did not impair the viability of target cells. T h e only exception
was 10.0 m M solution which depressed viability of lymphocytes f r o m the onset of
the oxidation reaction. By contrast with " 0 " h r oxidation, concentrationdependent cytotoxicity became evident in all 72 hr cell cultures. A t the same
time, the rates of [3H]-TdR incorporation by CrO3-oxidized cells invariably
decreased below control levels. E v e n at concentrations as low as 0.0005 m M , the
rates of [3H]-TdR incorporation by oxidized lymphocytes were inhibited.
Table 1. Comparative viability, [3H]-TdR incorporation and stimulation index (SI) of mouse
lymphocytes oxidized by f r O
Control
H5IO6
CrO3
1.0 mM
10.0 mM
1.0 mM
0.1 mM
0.01 mM
0.005 mM
0.001 mM
0.0005 mM
% Viability
0 hr
72 hr
96
82
65
100
96
100
97
97
ND
94
69
9
24
60
89
81
90
85
3
[3H]-TdR incorp.
SI
2727 + 290
19733 + 1255
93 + 88
380 + 57
835 + 173
1213 + 129
1930 5:104
2010 + 263
1783 5:71
1
7.23
0.03
0.14
0.31
0.44
0.71
0.73
0.65
Mouse lymphocytes (5 x 106/ml) were oxidized by f r O 3 at indicated concentrations for 20 min at 0~
Cells washed in PBS were cultured in RPMI-1640 medium as stated. During the last 16 h of culture,
the cells were pulsed with 1 # Ci[3H]-TdR, harvested and their radioactivity determined. Cells
oxidized by 1.0 mM H s I O 6 served as positive controls for oxidation-induced blastogenesis.
In addition to concentration-dependent cytotoxicity of c h r o m i u m on lymphocytes, oxidation time-dependent cytotoxicity was determined for the three
highest concentrations of CrO3 (Table 2). T h e results of these determinations
indicate that decrease in duration of the oxidation reaction from 20 to 2 min had
essentially no positive effect on subsequent viability of oxidized lymphocytes.
Similarly, the rate of [3H]-TdR incorporation by lymphocytes after 2 m i n
oxidation was below control levels (data not shown). T h e response of lymphocytes to tandem oxidation by CrO3 and by H5IO6, as well as to the reverse order
of oxidation reactions, are summarized in T a b l e 3.
The stimulation index and viability of lymphocytes initially oxidized by
H5IO6 and then oxidized again by CrO3 did not differ in any respect f r o m cells
oxidized only by CrO3 (Tables 1 and 3). If, however, the sequence of oxidation
reactions was reversed then the stimulation index, i.e. [3H]-TdR incorporation,
was invariably restored to normal (pre-oxidation) levels. Similarly the viability of
cells exposed to C r O 3 : H s I O 6 oxidation sequence tended to be higher than of the
cells oxidized solely by C r O 3.
Chromic Acid and Blastogenesis
Table 2.
351
Comparative viability of m o u s e lymphocytes oxidized by C r O 3 for 2 a n d 20 minutes
0 hr
Control
C r O 3 0.1 m M
2 min
1.0 m M
10.0 m M
Control
C r O 3 0.1 m M
20 min
1.0 m M
10.0 m M
24 hr
48 hr
72 hr
96 hr
94
99
93
84
86
84
60
41
82
77
62
40
65
66
33
12
65
34
11
6
100
100
100
65
95
71
95
43
87
59
69
47
81
63
31
9
68
37
6
0
Mouse lymphocytes (5 x 106/ml) were oxidized for 2 and 20 min by 1.0 m M C r O 3 at 0~
Oxidized cells were washed in PBS and cultured in RPMI-1640 m e d i u m as stated. Viability
determinations by trypan blue exclusion were performed at indicated intervals.
DISCUSSION
The results presented here can be summarized as follows: 1. Direct oxidation
of lymphocytes by Cr(VI) fails to trigger blastogenesis, but induces concentrationde,pendent cytotoxicity and inhibits [3H]-TdR incorporation. 2. Viability is
enhanced and [3H]-TdR incorporation by Cr(VI)-oxidized lymphocytes is restored to control levels after tandem oxidation by H 5 I O 6. 3. The reverse order of
oxidation does not nullify Cr(VI)-induced cytotoxicity or the inhibition of
[3H]-TdR incorporation. Oxidation of 1,2-diols by Cr(VI) can proceed in
accordance with two possible reaction schemes [13]. In its first variant, oxidation
of diols yields 0r
compounds, which may be oxidized further to
the corresponding carboxyls. Cleavage of carbon-carbon bond and the formation
of intermediate cyclic chromate esters comprises the second oxidation scheme. If
the first reaction was operative under stated conditions, then further oxidation of
0~-hydroxycarbonyls to the non-mitogenic carboxyls would effectively preclude
blastogenic response of oxidized lymphocytes. Similarly, no blastogenesis can be
expected after formation of membrane-bound cyclic chromate esters.
It was shown elsewhere [16-18] that Cr(VI) readily traverses plasma
Table 3.
CrO3
+
+
-
R e s p o n s e of m o u s e spleen cells to sequential oxidations by C r O 3 - H s I O 6 and by
HsIO6-CrO 3
Oxidation Sequence
H5IO 6
+
+
+
CrO 3
c p m + SD
+
2818
17457
238
3085
210
3H-TdR incorporation
S.I.
% viability
5:538
5:1810
5:143
5:472
5:150
1.00
6.19
0.08
1.09
0.08
81
71
31
44
26
Suspensions of 2.5 • 10 6 l y m p h o c y t e s / m l in PBS were oxidized by 1.0 m M C r O 3 for 20 min
at 0~ Oxidized cells were washed twice in PBS and oxidized again by 1.0 m M H s I O 6
under identical conditions. The reverse order of oxidation was also carried out identically.
Thereafter oxidized cells were cultured as was stated in Methods and their viability
determined at 72 h.
352
Malinin, Hornicek and Malinin
membrane, whereas Cr(III), a potent D N A cross-linking agent, does not [17, 18].
Unlike most in vitro studies of chromium interaction with cells, in this case
Cr(VI) interacted with lymphocytes directly at physiologically low temperature,
for no longer than 20 min, and in the absence of culture medium. Possible effect
on cells by the products of chromium reaction with culture medium was thereby
obviated and the amount of transmembrane Cr(VI) diffusion was either reduced
or abolished. These considerations and proven lability of cis-diol groups of
ribonucleotides to Cr(VI) oxidation [19] as well as demonstrable immobilization
of Cr(V) by cell membrane glycoproteins [20] jointly point to plasma membrane
diols as the initial site of Cr(VI) reduction by oxidized cells. The fact that Cr(III),
and presumably Cr(IV) and Cr(V) are readily oxidized by periodate [21]
prompted us to perform tandem oxidation of lymphocytes by Cr(VI) and by
1-15106. It was assumed that oxidation of membrane-bound chromium species by
H5IO6 would result in their displacement and solubilization in PBS, thus allowing
oxidized ceils to resume their function without chromium impediment. The
results, summarized in Table 3 seemingly vindicate our conjecture, and allow us
to conclude that during brief periods, chromic acid interacts primarily with
plasma membrane of lymphocytes.
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