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Toxic effect of chromium on cellular metabolism
The Science of the Total Environment, 71 (1988) 365-377 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
365
TOXIC EFFECT OF C H R O M I U M ON C E L L U L A R M E T A B O L I S M
P a t r i z i a D E B E T T O and Sisto LUCIANI D e p a r t m e n t o f Pharmacology, University of Padova, Largo E. Meneghetti 2, 3 5 1 3 1 P a d o v a - Italy
ABSTRACT The cytotoxic action of Cr(VI) in intact mammalian cells is strictly related to its c a r r i e r - m e d i a t e d transport across the plasma membrane and its reduction to Cr(III) inside the cell. A marked decrease in the ratio o f r e d u c e d / o x i d i z e d glutathione in rat thymocytes treated with d i c h r o m a t e indicates an i n v o l v e m e n t o f glutathione in the reduction of Cr(VI) to Cr(III) in these cells. Intracellular c h r o m i u m is shown to interfere w i t h specific steps of cellular energy m e t a b o l i s m in that it p r o d u c e s severe unbalance o f purine r i b o n u c l e o t i d e pools in hamster fibroblast cultures and inhibition of mitochondrial o x y g e n c o n s u m p t i o n in rat thymocytes at m i c r o m o l a r concentrations. To what extent the effects caused b y Cr(VI) are d i r e c t l y related to the reduction p r o c e s s or to the s u b s e q u e n t b i n d i n g of Cr(III), and p o s s i b l y of Cr(V), to biological molecules p l a y i n g a critical role in cell physiology, remains to be elucidated.
INTRODUCTION C h r o m i u m compounds have been shown to produce toxic
(ref. 1), mutagenic
(ref.2) and c a r c i n o g e n i c
(ref.3) effects in biological systems. The two oxida-
tion states of chromium,
Cr(VI)
and Cr(III), m a r k e d l y differ in their a c t i v i t y
in intact cells m a i n l y because of their d i f f e r e n t a b i l i t y to cross biological membranes. Cr(VI), as chromate,
readily permeates the cell membranes p o s s i b l y
through the general anion transport s y s t e m and sulphate anions, w h e r e a s Cr(III) through the p l a s m a m e m b r a n e
(refs.4-7) w h i c h transports phosphate
complexes are g e n e r a l l y unable to diffuse
(refs.8,9). The intracellular levels of Cr(III)
derive m a i n l y from the r e d u c t i o n o f Cr(VI), a l t h o u g h endocytosis could be a p o s s i b l e m e c h a n i s m of some Cr(III) other hand, Cr(III) ly than Cr(VI)
c o m p l e x uptake by the cells
(ref.2). On the
has b e e n found to a c c u m u l a t e on the cell surface more readi-
(ref. 10). The p r e s e n c e o f a v a r i e t y of c a t i o n - b i n d i n g sites in
the cell m e m b r a n e c o u l d a c c o u n t for this a c c u m u l a t i o n and for the e x t r e m e l y limited transport rate of Cr(III)
inside the cell
(ref. 10).
Due to its strong o x i d i z i n g power, chromate, once inside the cell, is rapidly r e d u c e d to Cr(III) w h i c h forms stable c o o r d i n a t i o n complexes w i t h several d i f f e r e n t ligands
(ref.8), thereby i n t e r f e r i n g with their p h y s i o l o g i c a l
tions. O w i n g to the stability of Cr(III) change rate with b i o l o g i c a l ligands
0048-9697/88/$03.50
func-
complexes and to their v e r y low ex-
(ref.8), the biological effects o f
© 1988' Elsevier Science Publishers B.V.
366 c h r o m i u m are thus strictly related to the endocellular site of Cr(VI) r e d u c t i o n to Cr(III) Cr(VI)
and to the subsequent b i n d i n g of Cr(III)
reduction can take place in d i f f e r e n t cell c o m p a r t m e n t s where elec-
trons are available systems,
to ligands within the cell.
from several biological r e d u c i n g molecules and enzymatic
such as cytosol, mitochondria, microsomes,
and nuclei
(refs.ll-15).
with the aim to elucidate the m o l e c u l a r m e c h a n i s m of c h r o m i u m toxic action at c e l l u l a r level, we investigated: o x i d a t i o n state;
(i) c h r o m i u m uptake and its intracellular
(2) effects of c h r o m i u m on the cellular energy metabolism;
(3) role of glutathione in cytoplasmic c h r o m i u m reduction. A c o m p r e h e n s i v e p i c t u r e of the data p r e s e n t e d in this paper is discussed.
EXPERImeNTAL Cells Cultures of the e s t a b l i s h e d p s e u d o d i p l o i d BHK 21 Syrian Hamster fibroblast line, grown as d e t a i l e d in
(ref. 16), were e m p l o y e d for n u c l e o t i d e pool composi-
tion studies. Suspensions o f rat thymocytes, p r e p a r e d as in (ref. 17), were used to study c h r o m i u m uptake and o x i d a t i o n state, and its effects o n cellular oxygen consumption.
C h r o m i u m uptake Chromate uptake in rat thymocytes was a s s a y e d by g a s - l i q u i d c h r o m a t o g r a p h i c (GLC) analysis of the c h r o m i u m intracellular levels. The GLC method, d e t a i l e d elsewhere
(P. Debetto et al., m a n u s c r i p t in preparation),
is based on the for-
mation of the volatile c h r o m i u m t r i f l u o r o - a c e t y l a c e t o n a t e by c h e l a t i o n - e x t r a c tion of Cr(III)
from aqueous solutions of acid d i g e s t e d samples with
l , l , l - t r i f l u o r o - 2 , 4 - p e n t a n e d i o n e in benzene.
C h r o m i u m o x i d a t i o n state The o x i d a t i o n state of intracellular c h r o m i u m was d e t e r m i n e d in rat thymocytes by electron paramagnetic resonance
(EPR) analysis. The E P R spectra were
run on a Bruker R 200 D spectrometer at 183 K, 1.6-G m o d u l a t i o n amplitude, 20 mW m i c r o w a v e power, 8.45 GHz microwave frequency and ixi06 gain
(P. A r s l a n
et al., m a n u s c r i p t in preparation).
E f f e c t s of c h r o m i u m on the cellular energy m e t a b o l i s m and role of ~ l u t a t h i o n e in cytoplasmic c h r o m i u m reduction The d e t e r m i n a t i o n of the composition of endogenous purine r i b o n u c l e o t i d e pools in hamster fibroblasts was accomplished by means of h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h i c (ref.18).
(HPLC) analysis following ~he procedure d e t a i l e d in
367 The m i t o c h o n d r i a l oxygen c o n s u m p t i o n in rat thymocytes was monitored with a Clark O 2 - e l e c t r o d e as d e s c r i b e d in
(ref.17).
The i n t r a c e l l u l a r levels o f glutathione pool c o m p o n e n t s were d e t e r m i n e d in rat thymocytes by HPLC analysis a c c o r d i n g to Reed et al.
(ref. 19).
Protein c o n c e n t r a t i o n was d e t e r m i n e d a c c o r d i n g to the method of Bensadoun and W e i n s t e i n
(ref.20), using bovine s e r u m albumin as the standard.
RESULTS AND D I S C U S S I O N Effects of c h r o m i u m on m e m b r a n e - b o u n d p r o t e i n s The cell m e m b r a n e is a possible target o f c h r o m i u m toxic action. In fact, Cr(VI)
is able to produce membrane functional damage by m o d i f y i n g membrane-
linked proteins,
as e x e m p l i f i e d by its action on the ectoenzyme M g 2 + - a c t i v a t e d
ATPase, whose a c t i v i t y has been c h a r a c t e r i z e d in plasma membranes of hamster fibroblasts by Cr(VI)
(BHK line)
(ref.21). Since the inhibition of the enzyme a c t i v i t y
was o b s e r v e d only upon p r e i n c u b a t i o n of the cells with d i c h r o m a t e
followed by its absence from the assay m e d i u m for Mg2+-ATPase activity,
this
i n h i b i t i o n has been a s c r i b e d to structural a l t e r a t i o n s caused by the o x i d i z i n g action o f Cr(VI) on the enzyme
(ref.21).
C h r o m i u m uptake and its i n t r a c e l l u l a r o x i d a t i o n state Kinetic a n a l y s i s o f c h r o m i u m uptake by rat thymocytes p e r f o r m e d by GLC indic a t e d that Cr(Vl)
anion was taken up by the cells d e p e n d i n g on the e x t r a c e l l u l a r
d i c h r o m a t e c o n c e n t r a t i o n and r e a c h e d intracellular levels in the m i l l i m o l a r range. C h r o m i u m uptake followed M i c h a e l i s - M e n t e n kinetics, carrier-mediated process
compatible with a
(ref.22).
C h r o m a t e uptake was completely inhibited by d i e t h y l m a l e i m i d e
(DEM)
(not
shown), as S H - b l o c k i n g agent. DEM could affect the r a p i d intracellular r e d u c t i o n of Cr(VI)
to Cr(III)
by i n h i b i t i n g g l u t a t h i o n e and/or other intracellular
reductants or i n a c t i v a t i n g the SH-groups of the chromate carrier. This finding suggests that the r e d u c t i o n of the h e x a v a l e n t c h r o m i u m m i g h t be the effective process r e s p o n s i b l e for the m a i n t e n a n c e of Cr(VI)
c o n c e n t r a t i o n gradient across
the p l a s m a membrane. The o x i d a t i o n state of i n t r a c e l l u l a r c h r o m i u m was d e t e r m i n e d by EPR analysis, w h i c h allows the selective assay of the p a r a m a g n e t i c species o f chromium, Cr(III)
and Cr(V), in whole cells
(P. Arslan et al., m a n u s c r i p t in preparation).
Thirty minutes i n c u b a t i o n of rat thymocytes w i t h 200 ~M K2Cr207 r e s u l t e d in the a p p e a r a n c e of an E P R signal w i t h g = 1.98
(ref.22), typical of paramagnetic
c h r o m i u m speciee Cr(III) and Cr(V), the latter b e i n g a well k n o w n i n t e r m e d i a t e of the m o n o e l e e t r o n i c r e d u c t i o n of HCrO4
(ref.23). The intensity of the signal
i n c r e a s e d w i t h the i n c r e a s e in the number o f cells and with the i n c u b a t i o n time. No E P R signal was d e t e r m i n e d upon incubation of the cells with Cr(III)
as
368 (A) o.-o AMP
r,--,.., ADP
Zk--A ATP
01 mM
05 mM
350-
350
o
o1
o:'°i- \ 50,
,
l
!'°°1
200-
\~'k, l
500
.o. 0
~)200.
050" l
153o oo 9o 12o
,
/.o ~
,
180
1 mM
i
,
50. w
i
153o 6o 90 12o
I~O
15~o o~ 9'0 I~O
i£
MINUTES OF TREATMENT (B)
o--o GMP
A~
m--m GDP
0.1raM
GTP
0.5 mM
lmM
350-
500t
~'°1 Ao
300-
450"],
450"
250-
250'
700
0
zo
0
o
150-
/
o
''L
100-
0j 150.
4" I
0
o
/
50' ~d'~W'~A
J~"-"-C~ 1530 60 90 120
180
400-
/
100. ~
50-
'~
i
150 q ~"'50-
\o
\
"'\&
100-
k
-------A
1530 60 90 120 180 MINUTES OF TREATMENT
I;~O 6'o ~ I~O I~o
Fig. i. V a r i a t i o n s o f e n d o g e n o u s (A) AMP, ADP, ATP and (B) GMP, GDP, GTP levels induced in BHK cells b y K2Cr207. T h e cell m o n o l a y e r s were treated w i t h 0.I - 1.0 m M K 2 C r 2 0 7 in BSS for 15 - 180 min. Values are e x p r e s s e d as p e r c e n t o f controls. M o d i f i e d from (ref. 16).
369 CrCl 3 (ref.22). Although Cr(VI),
the signal
was p r o d u c e d
it had d i f f e r e n t
from aqueous
solutions
amplitude
complex
Alterations Cr(VI)
reduction
of n u c l e o t i d e
has b e e n shown
fibroblasts
(BHK line)
relatively treated
incubation
to resume
composition controls
minimal
essential
specific
balanced
medium
interference
purine
fibroblasts
in the m e d i u m used
in BHK cells
of the treatment. Although
ATP,
(MEM)
significant In fact,
e v e n after
following
of c h r o m i u m
decrease
only with cells (BSS)
180 min re-
treatment
with nucleotide
in i n t r a c e l l u l a r
ribonucleotide
The p r e s e n c e
the d e p l e t i o n
salt solution
ATP,
the
pools was d e t e r m i n e d
treated w i t h i n c r e a s i n g
by means of H P L C analysis.
from the cells
(ref.24).
levels of e n d o g e n o u s
substrates.
in ATP c o n t e n t of Hamster
it was long-lasting. in Hanks'
that the
from the intra-
induced b y Cr(VI)
was q u a n t i t a t i v e l y
for the o b s e r v e d
of the e n d o g e n o u s
and hamster
dichromate
deriving
on the dose and d u r a t i o n
concentrations,
could account
It has thus been s u g g e s t e d or Cr(V),
could be c a l c u l a t e d
normal
in E a g l e ' s
to the signal o b t a i n e d
c o m p l e x e d w i t h biological
ATP i n d u c e d by Cr(VI)
h i g h Cr(VI)
of the cells w i t h
compared
to cause a m a r k e d d e c r e a s e
dependent
I). To test w h e t h e r
metabolism
TABLE
of Cr(VI),
w i t h 0.5 - 1.0 m M d i c h r o m a t e
were unable
(Table
to Cr(III)
pool c o m p o s i t i o n
An ID50 of 0.45 m M K 2 C r 2 0 7 of i n t r a c e l l u l a r
features
of CrCI 3 (ref.22).
E P R signal m i g h t be a t t r i b u t e d cellular
o n l y upon i n c u b a t i o n and
concentrations
of pool c o m p o n e n t s
for cell t r e a t m e n t s
in of
leaked
was also monitored.
1
Effects
of K 2 C r 2 0 7 p r e t r e a t m e n t
on i n t r a c e l l u l a r
ATP c o n t e n t
in BHK cells
The cell s u s p e n s i o n s were t r e a t e d with d i f f e r e n t c o n c e n t r a t i o n s of d i c h r o m a t e in BSS for 30 min. ATP concentration, e x p r e s s e d as nmoles A T P / m g of cell protein, w e r e d e t e r m i n e d at d i f f e r e n t times of r e i n c u b a t i o n in MEM. 30 min of preincubation in BSS (nmol ATP/mg)
Additions K2Cr207 (mM)
20.0 20.4 19.4 18.8 19.6
none 0. i0 0.25 0.50 1.00
Modified
from
in M E M after
treatment
0 min
60 m_in 120 min (nmol ATP/mg)
180 m i n
13.6 12.0 11.2 8.7 8.5
8.0 7.5 7.4 7.0 6.0
13.8 13.4 12.2 9.6 8.0
11.4 10.7 9.4 8.8 7.8
(ref.24).
All the tested doses of Cr(VI) levels,
accompanied
cursors
b o t h in the a d e n y l a t e
pectively),
Time of i n c u b a t i o n
of
produced
by a strong progressive
suggesting
a marked reduction increase
and the g u a n y l a t e
a~ i m p a i r m e n t
pool
of t r i p h o s p h a t e
in their n u c l e o t i d e (Fig.
of the p h o s p h o r y l a t i o n
i,
(A) and
pre-
(B), res-
steps r e s p o n s i b l e
370 of A T P and GTP production. by Cr(VI)
treatments
The severe u n b a l a n c e
was r e f l e c t e d
the value of its energy charge (ref.25)
TABLE
on the basis
of the a d e n y l a t e
system p r o d u c e d
by the dose- and t i m e - d e p e n d e n t
(Table 2), c a l c u l a t e d
according
decrease
in
to A t k i n s o n
of H P L C data.
2
Adenylate
energy
charge of c o n t r o l
and d i c h r o m a t e
treated
BHK cells
Values were c a l c u l a t e d on the basis of the a b s o l u t e n u c l e o t i d e c o n c e n t r a t i o n s d e t e r m i n e d by HPLC a n a l y s i s a c c o r d i n g to the equation of Atkinson: 1 ADP + 2ATP A M P + A D P + ATP Time of treatment min
Additions
30 60 90 120 180 Modified
from
The p a t t e r n o f v a r i a t i o n s
as a s i d e - e f f e c t
produced
syntheses
extracellular
amounts
T a k e n together,
plexes b e t w e e n
0.941 0.890 0.753 0.706 0.372
0.927 0.817 0.663 0.659 0.475
for the guanylate pool
However,
utilize
GTP,
conditions
by Cr(VI)
In fact,
these results
derived
substrates
GTP levels were less re-
namely
to the inhibition
R N A and p r o t e i n
through
the m e m b r a n e
in the plasma
medium
(Table
membrane
syn-
inosine
of the i n t e r f e r e n c e
could be did not and
3). The i n c r e a s e d
could be a s c r i b e d
that Cr(VI)
to their
by Cr(VI)
intra-
with
specifically
in ATP
of Cr(III)
com-
r e d u c t i o n of Cr(VI),
synthesis.
treated w i t h d i c h r o m a t e effects
interferes
of stable c o o r d i n a t i o n
from the i n t r a c e l l u l a r
from the i n h i b i t o r y
The (Table
complexes
lack o f r e c o v e r y i) could,
in
which a c c u m u l a t e d
intracellularly. Because regulators
of the c r i t i c a l
importance
of a great number
(B))
(A)),
(ref.26).
the formation
involved
levels in cells
i, panel
(ref. 18).
indicate
through
(Fig.
i, panel
o n l y hypoxanthine,
in the incubation
synthesis
pool
(Fig.
This can be a t t r i b u t e d
as a c o n s e q u e n c e
probably
Cr(III),
and/or ATP
result
0.966 0.935 0.906 0.863 0.791
of such p o o l components
accumulation,
on A T P synthesis,
fact,
induced
to nucleotides.
e a r l y steps of a d e n y l a t e
to c o n t r o l
0.977 0.975 0.971 0.966 0.915
which
treatment
c o u l d be d e t e c t e d
and enzymes
1.00
o f a leakage of t r i p h o s p h a t e s
since the d a m a g e
make it p e r m e a b l e
cellular
0.50
o f ATP depletion.
by these
The hypothesis
adenosine
0.i0
observed
than ATP levels.
of the m a c r o m o l e c u l a r
r u l e d out,
None
the same as for the a d e n y l a t e
duced by d i c h r o m a t e
thesis,
(mM)
(ref. 16).
was q u a l i t a t i v e l y probably
of K2Cr207
of n u c l e o t i d e s
of b i o s y n t h e t i c
in cell m e t a b o l i s m
pathways,
any perturbation
as of
371 TABLE
3
Variations
of p u r i n e
ribonucleotide
pool c o m p o s i t i o n
induced by K2Cr207
in BHK
cells The cells were t r e a t e d w i t h 0.5 m M d i c h r o m a t e in BSS for 60 min. The c o n c e n t r a tions of purine r i b o n u c l e o t i d e pool c o m p o n e n t s are e x p r e s s e d as p m o l e s / ~ g DNA. Additions
of
Intracellular
K2Cr207 (mM)
C o m p o n e n t s leaked in medium
pool c o m p o n e n t s
(pmol/~g DNA) (pmol/~g DNA) ATP 694 359
None 0.5
ADP 20.6 57.7
AMP 7.5 26.7
GTP 422 340
GDP 13.3 24.1
GMP 9.4
aHypo 136 479
bIno 9.1 46.7
CAdo 5.5 42.1
M o d i f i e d f r o m (ref. 18) . aHypo = h y p o x a n t h i n e bIno = inosine CAdo = adenosine
intracellular processes.
nucleotide
The o b s e r v e d
thus have a v a r i e t y
Effects
of c h r o m i u m
particularly
mainly maintained Incubation
oxygen
TABLE
suitable
to d i r e c t l y
for the normal
metabolism
functions
m~y
of the cell.
consumption consumption
for this
by o x i d a t i v e
study
phosphorylation
w i t h Cr(VI)
were m o n i t o r e d
since ATP
in a d o s e - d e p e n d e n t
level
manner
in r a t thymo-
in these cells
is
(ref.27).
as K 2 C r 2 0 7 d e c r e a s e d
p-trifluoromethoxyphenylhydrazone
consumption
a f f e c t n e a r l y all m e t a b o l i c
of c h r o m i u m w i t h n u c l e o t i d e
consequences
on o x y g e n
of t h y m o c y t e s
earbonyl-cyanide
is l i k e l y
interference
of severe
of c h r o m i u m on o x y g e n
The effects cytes,
pools
(FCCP)
the basal
stimulated
and
cellular
(Table 4). The effect o f c h r o m i u m
4
Effects
of K 2 C r 2 0 7
on c e l l u l a r
oxygen consumption
in r a t t h y m o c y t e s
50xi06 c e l l s / m l w e r e t r e a t e d w i t h 0.12 - 1.00 m M K 2 C r 2 0 7 in BSS for I0 min. W h e n present, FCCP was 0.4 ~M. Data are means o f at least 3 d i f f e r e n t experim e n t s ± S.D. A d d i t i o n s of K 2 C r 2 0 7 (mM)
Basal
None 0.12 0.25 0.50 1.00 Modified
02 c o n s u m p t i o n FCCP s t i m u l a t e d (natoms/min/106 cells)
0.41 0.19 0.17 0.16 0.15 from
(ref.17).
-+ i _+ + +
0.05 0.02 0.02 0.03 0.03
0.76 0.63 0.33 0.Ii 0.ii
+ + -+ -+ +
02 c o n s u m p t i o n
0.06 0.05 0.02 0.02 0.02
372 was evident on 02 c o n s u m p t i o n stimulated either by FCCP or ADP
(ref. 17),
s u g g e s t i n g a direct inhibition of electron flow in the r e s p i r a t o r y chain by Cr(VI) rather than of energy transfer. Cr(III) on 02 c o n s u m p t i o n
When the effects of Cr(VI) "in situ"
mitochondria
as CrCI 3 did not have any effect
(ref. 17). and Cr(III)
were d i r e c t l y tested on thymocyte
(digitonin p e r m e a b i l i z e d thymocytes),
it was evident
that the d o s e - d e p e n d e n t i n h i b i t i o n of the maximal 02 c o n s u m p t i o n rate by c h r o m i u m d e p e n d e d on the o x i d i z a b l e substrate used succinate,
a flavin adenine d i n u c l e o t i d e
100-~
(A)
(Fig. 2,
(A) and
(FAD)-linked substrate,
100
80.
80
60,
60
(B)). With
and in the
(B)
o
o
o 40
40-
20-
20-
120 250 500 K2Cr20 7 (HM)
1~)00
120 250
500 CrCI 3 (~M)
lPoo
Fig. 2. E f f e c t s of K 2 C r 2 0 7 and CrC13 on maximal 02 c o n s u m p t i o n rate of digitonin treated rat thymocytes. 50x106 cells/ml were treated with 2-i,000 ~M K 2 C r 2 0 7 (A) and w i t h 120-1,000 ~M CrCI 3 (B) in BSS for 10 m_in. After 5 min at 30oc, 50 HM d i g i t o n i n and 5 uM cytochrome C were added. The m e d i u m was supplemented w i t h 2 m M glutamate and 2 m M malate (e-e) or with i0 m M succinate and 2 pM rotenone (o-o). FCCP was 0.4 uM and was p r e c e d e d by the a d d i t i o n of 1 ~g oligomycin/ml. Modified from (ref.17).
p r e s e n c e of rotenone w h i c h causes the c o m p l e x i to be totally reduced,
50% inhi-
bition o f FCCP stimulated 02 c o n s u m p t i o n was o b t a i n e d w i t h 400 ~M Cr(VI) and 200 ~M Cr(III); while u s i n g glutamate p l u s malate, n i c o t i n a m i n e a d e n i n e dinucleotide
(NAD+)-linked substrates,
50% inhibition was o b t a i n e d with 20 uM
Cr(VI) and 400 pM Cr(III). Q u a l i t a t i v e l y similar results were o b t a i n e d in isolated rat liver m i t o c h o n d r i a
(ref.17).
These findings indicate that the inhibition of the mitochondrial r e s p i r a t i o n by Cr(VI)
is more severe when N A D - l i n k e d substrates are oxidized,
concentrations
373 o f Cr(VI)
in the low m i c r o m o l a r range b e i n g active. These results are in good
a g r e e m e n t w i t h those r e p o r t e d by Ryberg and Alexander
(ref.28)
for the inhibi-
tory action of chromate on 02 c o n s u m p t i o n in isolated liver mitochondria. The molecular m e c h a n i s m of Cr(VI)
and Cr(III)
p h o s p h o r y l a t i o n remains to be established:
inhibition of oxidative
Cr(VI) m i g h t accept electrons de-
r i v i n g m a i n l y from N A D - l i n k e d substrates at the level of c o m p l e x e l e c t r o n t r a n s p o r t chain,
1 of the
thus i n h i b i t i n g o x y g e n r e d u c t i o n and/or o x i d i z e
essential components of the r e s p i r a t o r y chain; Cr(III), d e r i v e d from Cr(VI) reduction, m i g h t form c o o r d i n a t i o n complexes w i t h such components involved in e l e c t r o n transport. Since NADH m i t o c h o n d r i a l pool r e p r e s e n t s the p r i m a r y e n d o g e n o u s electron source in the cell,
the inhibition of m i t o c h o n d r i a l r e s p i r a t i o n by c h r o m i u m
may be e x p e c t e d to have great c o n s e q u e n c e s
Glutathione
in c y t o p l a s m i c Cr(VI)
for the cell physiology.
reduction
Given that a key step in the toxic action of Cr(VI) r e d u c t i o n to Cr(III),
is its intracellular
the v a r i a t i o n s of g l u t a t h i o n e pool c o m p o s i t i o n induced in
rat thymocytes by treatment with d i c h r o m a t e have been d e t e r m i n e d by means of HPLC a n a l y s i s
(ref. 19). Glutathione
(GSH) is indeed the main cytosolic r e d u c t a n t
and it has been r e p o r t e d to n o n - e n z y m a t i c a l l y reduce Cr(VI) s i g n i f i c a n t rate under conditions of p h y s i o l o g i c a l pH
to Cr(III) at a
(ref. 13).
Rat thymocytes have been found to c o n t a i n 2.2 - 4.6 m M GSH and 53 - 100 ~M glutathione disulphide cells
(GSSG), a s s u m i n g an intracellular volume of 0. i ~i/i06
(ref.29).
Table 5 shows that treatments of thymocytes with d i c h r o m a t e induced a strong
TABLE 5 V a r i a t i o n s of glutathione pool c o m p o s i t i o n i n d u c e d in rat thymycytes by K 2 C r 2 0 7 The cells were treated w i t h 1.0 - 2.0 m M K 2 C r 2 0 7 in BSS for 60 and 120 min. The c o n c e n t r a t i o n s of g l u t a t h i o n e pool c o m p o n e n t s are e x p r e s s e d as n m o l e s / m g of cell protein. Time of treatment (min) 60
120
Additions of K 2 C r 2 0 7 (mM)
GSH
None 1.0 2.0 None 2.0
3.2 2.8 2.4 7.4 4.2
aGlut = glutamate bAspart = aspartate
GSSG
GSH/GSSG
aGlut
bAspart
Glut + Aspart
21.3 10.8 8.0 53.0 27.7
32.6 30.2 29.4 87.6 76.1
(nmol/mg prot) 0.09 0.14 0.22 0.13 0.41
35 20 ii 57 I0
11.3 19.4 21.4 34.6 48.4
374 decrease in the ratio of r e d u c e d / o x i d i z e d glutathione, d e p e n d e n t on the dose and d u r a t i o n of exposure,
suggesting an involvement of GSH in the intracellular
reduction p r o c e s s of Cr(VI)
to Cr(III)
in these cells. Furthermore,
a marked
increase in the intracellular glutamate levels accompanied by a p a r a l l e l decrease in aspartate levels was o b s e r v e d
(Table 5). The intracellular accumula-
tion of glutamate could be due to the inhibition of its m i t o c h o n d r i a l c a t a b o l i s m by Cr(VI), which decreases the a v a i l a b i l i t y of the o x i d i z e d nicotinamide adenine dinucleotides by i n t e r a c t i n g at the level of c o m p l e x i of the electron transport chain.
CONCLUSIONS A summary scheme of major events involved in the p r o d u c t i o n of cytotoxic effects b y c h r o m i u m is p r e s e n t e d in Fig.
co% o%ds-- c§IxeE2DO0 ' --OS--o%C! -- -ODI Cr (Vl) compounds~
H
~1 -MEDIATED .... TRANSPORTH L;ru4
~
3.
-.)I-0/_~..~/,CD
:r. . . . ' bru4
/2crC~llic)omplePxe ~ ~ DNA-'PROTEIN U~72_ ~i I~R~ ~ L'rU4 ~ ,-; ~'!1 n
..
ADPJ
Fig. 3. S u m m a r y scheme of major events involved in c h r o m i u m toxic action at c e l l u l a r level. M o d i f i e d from: A.G. Levis and V. Bianchi, in S. Langard (Ed.), Biological and Environmental Aspects of Chromium, Elsevier, N o r t h - H o l l a n d Biomedical Press, Amsterdam, 1982, p.171.
375 A l t h o u g h Cr(VI)
has been p r o v e d to p r o d u c e membrane
m o d i f y i n g m e m b r a n e - l i n k e d proteins
(e.g. Mg2+-ATPase),
functional damage by its cytotoxic action
seems to be m a i n l y c o n f i n e d at intracellular level. Given that the transport of Cr(III) Cr(VI)
complexes p o s s i b l y by endocytosis is e x t r e m l y limited, e x t r a c e l l u l a r r e d u c t i o n by a variety o f systems,
including serum proteins,
acid and c o m p o n e n t s in the outer surface of the cell membrane, i m p o r t a n t m e c h a n i s m of c h r o m i u m detoxification. Therefore,
ascorbic
represents an
the a b i l i t y o f Cr(VI)
to produce cytotoxic e f f e c t s is strictly related to its facilitated transport across the p l a s m a m e m b r a n e and its reduction to Cr(III) sibly t h r o u g h the intermediate reactive Cr(V) w i t h i n the c y t o p l a s m Cr(VI) cysteine,
is reduced in : (I) cytosol,
ascorbate and D T - d i a p h o r a s e
NAD(P) H - d e p e n d e n t flavoprotein for the process;
inside the cell, pos-
form.
(ref. 15) -
(2) microsomes,
P 4 5 0 - r e d u c t a s e e n z y m a t i c system;
where GSH but also
- the latter b e i n g a F A D - c o n t a i n i n g are a m o n g the m o l e c u l e s responsible
mainly by the NAD(P) H - d e p e n d e n t cytochrome (3) mitochondria,
where Cr(VI)
m i g h t accept
e l e c t r o n s d e r i v i n g m a i n l y from N A D - l i n k e d s u b s t r a t e s at the level of c o m p l e x 1 of the e l e c t r o n transport chain;
(4) nuclei.
I n t r a c e l l u l a r c h r o m i u m has been shown to interfere with specific steps of c e l l u l a r energy m e t a b o l i s m in that it p r o d u c e s severe unbalance of purine ribonucleotide p o o l s and inhibition of m i t o c h o n d r i a l o x y g e n consumption. However,
it is not yet k n o w n to w h a t extent the effects caused by Cr(VI)
are d i r e c t l y related to the r e d u c t i o n p r o c e s s or to the subsequent binding of Cr(III)
and p o s s i b l y o f Cr(V)
to biological molecules w h i c h p l a y a critical
role in cell physiology.
ACKNOWLEDGEMENTS This work was supported b y grants from the N a t i o n a l R e s e a r c h Council of Italy
(CNR, P r o g e t t o F i n a l i z z a t o Medicina P r e v e n t i v a e Riabilitativa,
p r o g e t t o Rischio Tossicologico,
C R 83.02707.56)
and the V e n e t i a Region
Sotto(Centro
Regionale di Alta S p e c i a l i z z a z i o n e in C a n c e r o g e n e s i Ambientale). We a c k n o w l e d g e P. Arslan,
M. Beltrame and T. Pozzan for m a k i n g a v a i l a b l e
u n p u b l i s h e d o b s e r v a t i o n s on EPR.
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2 3 4
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365
TOXIC EFFECT OF C H R O M I U M ON C E L L U L A R M E T A B O L I S M
P a t r i z i a D E B E T T O and Sisto LUCIANI D e p a r t m e n t o f Pharmacology, University of Padova, Largo E. Meneghetti 2, 3 5 1 3 1 P a d o v a - Italy
ABSTRACT The cytotoxic action of Cr(VI) in intact mammalian cells is strictly related to its c a r r i e r - m e d i a t e d transport across the plasma membrane and its reduction to Cr(III) inside the cell. A marked decrease in the ratio o f r e d u c e d / o x i d i z e d glutathione in rat thymocytes treated with d i c h r o m a t e indicates an i n v o l v e m e n t o f glutathione in the reduction of Cr(VI) to Cr(III) in these cells. Intracellular c h r o m i u m is shown to interfere w i t h specific steps of cellular energy m e t a b o l i s m in that it p r o d u c e s severe unbalance o f purine r i b o n u c l e o t i d e pools in hamster fibroblast cultures and inhibition of mitochondrial o x y g e n c o n s u m p t i o n in rat thymocytes at m i c r o m o l a r concentrations. To what extent the effects caused b y Cr(VI) are d i r e c t l y related to the reduction p r o c e s s or to the s u b s e q u e n t b i n d i n g of Cr(III), and p o s s i b l y of Cr(V), to biological molecules p l a y i n g a critical role in cell physiology, remains to be elucidated.
INTRODUCTION C h r o m i u m compounds have been shown to produce toxic
(ref. 1), mutagenic
(ref.2) and c a r c i n o g e n i c
(ref.3) effects in biological systems. The two oxida-
tion states of chromium,
Cr(VI)
and Cr(III), m a r k e d l y differ in their a c t i v i t y
in intact cells m a i n l y because of their d i f f e r e n t a b i l i t y to cross biological membranes. Cr(VI), as chromate,
readily permeates the cell membranes p o s s i b l y
through the general anion transport s y s t e m and sulphate anions, w h e r e a s Cr(III) through the p l a s m a m e m b r a n e
(refs.4-7) w h i c h transports phosphate
complexes are g e n e r a l l y unable to diffuse
(refs.8,9). The intracellular levels of Cr(III)
derive m a i n l y from the r e d u c t i o n o f Cr(VI), a l t h o u g h endocytosis could be a p o s s i b l e m e c h a n i s m of some Cr(III) other hand, Cr(III) ly than Cr(VI)
c o m p l e x uptake by the cells
(ref.2). On the
has b e e n found to a c c u m u l a t e on the cell surface more readi-
(ref. 10). The p r e s e n c e o f a v a r i e t y of c a t i o n - b i n d i n g sites in
the cell m e m b r a n e c o u l d a c c o u n t for this a c c u m u l a t i o n and for the e x t r e m e l y limited transport rate of Cr(III)
inside the cell
(ref. 10).
Due to its strong o x i d i z i n g power, chromate, once inside the cell, is rapidly r e d u c e d to Cr(III) w h i c h forms stable c o o r d i n a t i o n complexes w i t h several d i f f e r e n t ligands
(ref.8), thereby i n t e r f e r i n g with their p h y s i o l o g i c a l
tions. O w i n g to the stability of Cr(III) change rate with b i o l o g i c a l ligands
0048-9697/88/$03.50
func-
complexes and to their v e r y low ex-
(ref.8), the biological effects o f
© 1988' Elsevier Science Publishers B.V.
366 c h r o m i u m are thus strictly related to the endocellular site of Cr(VI) r e d u c t i o n to Cr(III) Cr(VI)
and to the subsequent b i n d i n g of Cr(III)
reduction can take place in d i f f e r e n t cell c o m p a r t m e n t s where elec-
trons are available systems,
to ligands within the cell.
from several biological r e d u c i n g molecules and enzymatic
such as cytosol, mitochondria, microsomes,
and nuclei
(refs.ll-15).
with the aim to elucidate the m o l e c u l a r m e c h a n i s m of c h r o m i u m toxic action at c e l l u l a r level, we investigated: o x i d a t i o n state;
(i) c h r o m i u m uptake and its intracellular
(2) effects of c h r o m i u m on the cellular energy metabolism;
(3) role of glutathione in cytoplasmic c h r o m i u m reduction. A c o m p r e h e n s i v e p i c t u r e of the data p r e s e n t e d in this paper is discussed.
EXPERImeNTAL Cells Cultures of the e s t a b l i s h e d p s e u d o d i p l o i d BHK 21 Syrian Hamster fibroblast line, grown as d e t a i l e d in
(ref. 16), were e m p l o y e d for n u c l e o t i d e pool composi-
tion studies. Suspensions o f rat thymocytes, p r e p a r e d as in (ref. 17), were used to study c h r o m i u m uptake and o x i d a t i o n state, and its effects o n cellular oxygen consumption.
C h r o m i u m uptake Chromate uptake in rat thymocytes was a s s a y e d by g a s - l i q u i d c h r o m a t o g r a p h i c (GLC) analysis of the c h r o m i u m intracellular levels. The GLC method, d e t a i l e d elsewhere
(P. Debetto et al., m a n u s c r i p t in preparation),
is based on the for-
mation of the volatile c h r o m i u m t r i f l u o r o - a c e t y l a c e t o n a t e by c h e l a t i o n - e x t r a c tion of Cr(III)
from aqueous solutions of acid d i g e s t e d samples with
l , l , l - t r i f l u o r o - 2 , 4 - p e n t a n e d i o n e in benzene.
C h r o m i u m o x i d a t i o n state The o x i d a t i o n state of intracellular c h r o m i u m was d e t e r m i n e d in rat thymocytes by electron paramagnetic resonance
(EPR) analysis. The E P R spectra were
run on a Bruker R 200 D spectrometer at 183 K, 1.6-G m o d u l a t i o n amplitude, 20 mW m i c r o w a v e power, 8.45 GHz microwave frequency and ixi06 gain
(P. A r s l a n
et al., m a n u s c r i p t in preparation).
E f f e c t s of c h r o m i u m on the cellular energy m e t a b o l i s m and role of ~ l u t a t h i o n e in cytoplasmic c h r o m i u m reduction The d e t e r m i n a t i o n of the composition of endogenous purine r i b o n u c l e o t i d e pools in hamster fibroblasts was accomplished by means of h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h i c (ref.18).
(HPLC) analysis following ~he procedure d e t a i l e d in
367 The m i t o c h o n d r i a l oxygen c o n s u m p t i o n in rat thymocytes was monitored with a Clark O 2 - e l e c t r o d e as d e s c r i b e d in
(ref.17).
The i n t r a c e l l u l a r levels o f glutathione pool c o m p o n e n t s were d e t e r m i n e d in rat thymocytes by HPLC analysis a c c o r d i n g to Reed et al.
(ref. 19).
Protein c o n c e n t r a t i o n was d e t e r m i n e d a c c o r d i n g to the method of Bensadoun and W e i n s t e i n
(ref.20), using bovine s e r u m albumin as the standard.
RESULTS AND D I S C U S S I O N Effects of c h r o m i u m on m e m b r a n e - b o u n d p r o t e i n s The cell m e m b r a n e is a possible target o f c h r o m i u m toxic action. In fact, Cr(VI)
is able to produce membrane functional damage by m o d i f y i n g membrane-
linked proteins,
as e x e m p l i f i e d by its action on the ectoenzyme M g 2 + - a c t i v a t e d
ATPase, whose a c t i v i t y has been c h a r a c t e r i z e d in plasma membranes of hamster fibroblasts by Cr(VI)
(BHK line)
(ref.21). Since the inhibition of the enzyme a c t i v i t y
was o b s e r v e d only upon p r e i n c u b a t i o n of the cells with d i c h r o m a t e
followed by its absence from the assay m e d i u m for Mg2+-ATPase activity,
this
i n h i b i t i o n has been a s c r i b e d to structural a l t e r a t i o n s caused by the o x i d i z i n g action o f Cr(VI) on the enzyme
(ref.21).
C h r o m i u m uptake and its i n t r a c e l l u l a r o x i d a t i o n state Kinetic a n a l y s i s o f c h r o m i u m uptake by rat thymocytes p e r f o r m e d by GLC indic a t e d that Cr(Vl)
anion was taken up by the cells d e p e n d i n g on the e x t r a c e l l u l a r
d i c h r o m a t e c o n c e n t r a t i o n and r e a c h e d intracellular levels in the m i l l i m o l a r range. C h r o m i u m uptake followed M i c h a e l i s - M e n t e n kinetics, carrier-mediated process
compatible with a
(ref.22).
C h r o m a t e uptake was completely inhibited by d i e t h y l m a l e i m i d e
(DEM)
(not
shown), as S H - b l o c k i n g agent. DEM could affect the r a p i d intracellular r e d u c t i o n of Cr(VI)
to Cr(III)
by i n h i b i t i n g g l u t a t h i o n e and/or other intracellular
reductants or i n a c t i v a t i n g the SH-groups of the chromate carrier. This finding suggests that the r e d u c t i o n of the h e x a v a l e n t c h r o m i u m m i g h t be the effective process r e s p o n s i b l e for the m a i n t e n a n c e of Cr(VI)
c o n c e n t r a t i o n gradient across
the p l a s m a membrane. The o x i d a t i o n state of i n t r a c e l l u l a r c h r o m i u m was d e t e r m i n e d by EPR analysis, w h i c h allows the selective assay of the p a r a m a g n e t i c species o f chromium, Cr(III)
and Cr(V), in whole cells
(P. Arslan et al., m a n u s c r i p t in preparation).
Thirty minutes i n c u b a t i o n of rat thymocytes w i t h 200 ~M K2Cr207 r e s u l t e d in the a p p e a r a n c e of an E P R signal w i t h g = 1.98
(ref.22), typical of paramagnetic
c h r o m i u m speciee Cr(III) and Cr(V), the latter b e i n g a well k n o w n i n t e r m e d i a t e of the m o n o e l e e t r o n i c r e d u c t i o n of HCrO4
(ref.23). The intensity of the signal
i n c r e a s e d w i t h the i n c r e a s e in the number o f cells and with the i n c u b a t i o n time. No E P R signal was d e t e r m i n e d upon incubation of the cells with Cr(III)
as
368 (A) o.-o AMP
r,--,.., ADP
Zk--A ATP
01 mM
05 mM
350-
350
o
o1
o:'°i- \ 50,
,
l
!'°°1
200-
\~'k, l
500
.o. 0
~)200.
050" l
153o oo 9o 12o
,
/.o ~
,
180
1 mM
i
,
50. w
i
153o 6o 90 12o
I~O
15~o o~ 9'0 I~O
i£
MINUTES OF TREATMENT (B)
o--o GMP
A~
m--m GDP
0.1raM
GTP
0.5 mM
lmM
350-
500t
~'°1 Ao
300-
450"],
450"
250-
250'
700
0
zo
0
o
150-
/
o
''L
100-
0j 150.
4" I
0
o
/
50' ~d'~W'~A
J~"-"-C~ 1530 60 90 120
180
400-
/
100. ~
50-
'~
i
150 q ~"'50-
\o
\
"'\&
100-
k
-------A
1530 60 90 120 180 MINUTES OF TREATMENT
I;~O 6'o ~ I~O I~o
Fig. i. V a r i a t i o n s o f e n d o g e n o u s (A) AMP, ADP, ATP and (B) GMP, GDP, GTP levels induced in BHK cells b y K2Cr207. T h e cell m o n o l a y e r s were treated w i t h 0.I - 1.0 m M K 2 C r 2 0 7 in BSS for 15 - 180 min. Values are e x p r e s s e d as p e r c e n t o f controls. M o d i f i e d from (ref. 16).
369 CrCl 3 (ref.22). Although Cr(VI),
the signal
was p r o d u c e d
it had d i f f e r e n t
from aqueous
solutions
amplitude
complex
Alterations Cr(VI)
reduction
of n u c l e o t i d e
has b e e n shown
fibroblasts
(BHK line)
relatively treated
incubation
to resume
composition controls
minimal
essential
specific
balanced
medium
interference
purine
fibroblasts
in the m e d i u m used
in BHK cells
of the treatment. Although
ATP,
(MEM)
significant In fact,
e v e n after
following
of c h r o m i u m
decrease
only with cells (BSS)
180 min re-
treatment
with nucleotide
in i n t r a c e l l u l a r
ribonucleotide
The p r e s e n c e
the d e p l e t i o n
salt solution
ATP,
the
pools was d e t e r m i n e d
treated w i t h i n c r e a s i n g
by means of H P L C analysis.
from the cells
(ref.24).
levels of e n d o g e n o u s
substrates.
in ATP c o n t e n t of Hamster
it was long-lasting. in Hanks'
that the
from the intra-
induced b y Cr(VI)
was q u a n t i t a t i v e l y
for the o b s e r v e d
of the e n d o g e n o u s
and hamster
dichromate
deriving
on the dose and d u r a t i o n
concentrations,
could account
It has thus been s u g g e s t e d or Cr(V),
could be c a l c u l a t e d
normal
in E a g l e ' s
to the signal o b t a i n e d
c o m p l e x e d w i t h biological
ATP i n d u c e d by Cr(VI)
h i g h Cr(VI)
of the cells w i t h
compared
to cause a m a r k e d d e c r e a s e
dependent
I). To test w h e t h e r
metabolism
TABLE
of Cr(VI),
w i t h 0.5 - 1.0 m M d i c h r o m a t e
were unable
(Table
to Cr(III)
pool c o m p o s i t i o n
An ID50 of 0.45 m M K 2 C r 2 0 7 of i n t r a c e l l u l a r
features
of CrCI 3 (ref.22).
E P R signal m i g h t be a t t r i b u t e d cellular
o n l y upon i n c u b a t i o n and
concentrations
of pool c o m p o n e n t s
for cell t r e a t m e n t s
in of
leaked
was also monitored.
1
Effects
of K 2 C r 2 0 7 p r e t r e a t m e n t
on i n t r a c e l l u l a r
ATP c o n t e n t
in BHK cells
The cell s u s p e n s i o n s were t r e a t e d with d i f f e r e n t c o n c e n t r a t i o n s of d i c h r o m a t e in BSS for 30 min. ATP concentration, e x p r e s s e d as nmoles A T P / m g of cell protein, w e r e d e t e r m i n e d at d i f f e r e n t times of r e i n c u b a t i o n in MEM. 30 min of preincubation in BSS (nmol ATP/mg)
Additions K2Cr207 (mM)
20.0 20.4 19.4 18.8 19.6
none 0. i0 0.25 0.50 1.00
Modified
from
in M E M after
treatment
0 min
60 m_in 120 min (nmol ATP/mg)
180 m i n
13.6 12.0 11.2 8.7 8.5
8.0 7.5 7.4 7.0 6.0
13.8 13.4 12.2 9.6 8.0
11.4 10.7 9.4 8.8 7.8
(ref.24).
All the tested doses of Cr(VI) levels,
accompanied
cursors
b o t h in the a d e n y l a t e
pectively),
Time of i n c u b a t i o n
of
produced
by a strong progressive
suggesting
a marked reduction increase
and the g u a n y l a t e
a~ i m p a i r m e n t
pool
of t r i p h o s p h a t e
in their n u c l e o t i d e (Fig.
of the p h o s p h o r y l a t i o n
i,
(A) and
pre-
(B), res-
steps r e s p o n s i b l e
370 of A T P and GTP production. by Cr(VI)
treatments
The severe u n b a l a n c e
was r e f l e c t e d
the value of its energy charge (ref.25)
TABLE
on the basis
of the a d e n y l a t e
system p r o d u c e d
by the dose- and t i m e - d e p e n d e n t
(Table 2), c a l c u l a t e d
according
decrease
in
to A t k i n s o n
of H P L C data.
2
Adenylate
energy
charge of c o n t r o l
and d i c h r o m a t e
treated
BHK cells
Values were c a l c u l a t e d on the basis of the a b s o l u t e n u c l e o t i d e c o n c e n t r a t i o n s d e t e r m i n e d by HPLC a n a l y s i s a c c o r d i n g to the equation of Atkinson: 1 ADP + 2ATP A M P + A D P + ATP Time of treatment min
Additions
30 60 90 120 180 Modified
from
The p a t t e r n o f v a r i a t i o n s
as a s i d e - e f f e c t
produced
syntheses
extracellular
amounts
T a k e n together,
plexes b e t w e e n
0.941 0.890 0.753 0.706 0.372
0.927 0.817 0.663 0.659 0.475
for the guanylate pool
However,
utilize
GTP,
conditions
by Cr(VI)
In fact,
these results
derived
substrates
GTP levels were less re-
namely
to the inhibition
R N A and p r o t e i n
through
the m e m b r a n e
in the plasma
medium
(Table
membrane
syn-
inosine
of the i n t e r f e r e n c e
could be did not and
3). The i n c r e a s e d
could be a s c r i b e d
that Cr(VI)
to their
by Cr(VI)
intra-
with
specifically
in ATP
of Cr(III)
com-
r e d u c t i o n of Cr(VI),
synthesis.
treated w i t h d i c h r o m a t e effects
interferes
of stable c o o r d i n a t i o n
from the i n t r a c e l l u l a r
from the i n h i b i t o r y
The (Table
complexes
lack o f r e c o v e r y i) could,
in
which a c c u m u l a t e d
intracellularly. Because regulators
of the c r i t i c a l
importance
of a great number
(B))
(A)),
(ref.26).
the formation
involved
levels in cells
i, panel
(ref. 18).
indicate
through
(Fig.
i, panel
o n l y hypoxanthine,
in the incubation
synthesis
pool
(Fig.
This can be a t t r i b u t e d
as a c o n s e q u e n c e
probably
Cr(III),
and/or ATP
result
0.966 0.935 0.906 0.863 0.791
of such p o o l components
accumulation,
on A T P synthesis,
fact,
induced
to nucleotides.
e a r l y steps of a d e n y l a t e
to c o n t r o l
0.977 0.975 0.971 0.966 0.915
which
treatment
c o u l d be d e t e c t e d
and enzymes
1.00
o f a leakage of t r i p h o s p h a t e s
since the d a m a g e
make it p e r m e a b l e
cellular
0.50
o f ATP depletion.
by these
The hypothesis
adenosine
0.i0
observed
than ATP levels.
of the m a c r o m o l e c u l a r
r u l e d out,
None
the same as for the a d e n y l a t e
duced by d i c h r o m a t e
thesis,
(mM)
(ref. 16).
was q u a l i t a t i v e l y probably
of K2Cr207
of n u c l e o t i d e s
of b i o s y n t h e t i c
in cell m e t a b o l i s m
pathways,
any perturbation
as of
371 TABLE
3
Variations
of p u r i n e
ribonucleotide
pool c o m p o s i t i o n
induced by K2Cr207
in BHK
cells The cells were t r e a t e d w i t h 0.5 m M d i c h r o m a t e in BSS for 60 min. The c o n c e n t r a tions of purine r i b o n u c l e o t i d e pool c o m p o n e n t s are e x p r e s s e d as p m o l e s / ~ g DNA. Additions
of
Intracellular
K2Cr207 (mM)
C o m p o n e n t s leaked in medium
pool c o m p o n e n t s
(pmol/~g DNA) (pmol/~g DNA) ATP 694 359
None 0.5
ADP 20.6 57.7
AMP 7.5 26.7
GTP 422 340
GDP 13.3 24.1
GMP 9.4
aHypo 136 479
bIno 9.1 46.7
CAdo 5.5 42.1
M o d i f i e d f r o m (ref. 18) . aHypo = h y p o x a n t h i n e bIno = inosine CAdo = adenosine
intracellular processes.
nucleotide
The o b s e r v e d
thus have a v a r i e t y
Effects
of c h r o m i u m
particularly
mainly maintained Incubation
oxygen
TABLE
suitable
to d i r e c t l y
for the normal
metabolism
functions
m~y
of the cell.
consumption consumption
for this
by o x i d a t i v e
study
phosphorylation
w i t h Cr(VI)
were m o n i t o r e d
since ATP
in a d o s e - d e p e n d e n t
level
manner
in r a t thymo-
in these cells
is
(ref.27).
as K 2 C r 2 0 7 d e c r e a s e d
p-trifluoromethoxyphenylhydrazone
consumption
a f f e c t n e a r l y all m e t a b o l i c
of c h r o m i u m w i t h n u c l e o t i d e
consequences
on o x y g e n
of t h y m o c y t e s
earbonyl-cyanide
is l i k e l y
interference
of severe
of c h r o m i u m on o x y g e n
The effects cytes,
pools
(FCCP)
the basal
stimulated
and
cellular
(Table 4). The effect o f c h r o m i u m
4
Effects
of K 2 C r 2 0 7
on c e l l u l a r
oxygen consumption
in r a t t h y m o c y t e s
50xi06 c e l l s / m l w e r e t r e a t e d w i t h 0.12 - 1.00 m M K 2 C r 2 0 7 in BSS for I0 min. W h e n present, FCCP was 0.4 ~M. Data are means o f at least 3 d i f f e r e n t experim e n t s ± S.D. A d d i t i o n s of K 2 C r 2 0 7 (mM)
Basal
None 0.12 0.25 0.50 1.00 Modified
02 c o n s u m p t i o n FCCP s t i m u l a t e d (natoms/min/106 cells)
0.41 0.19 0.17 0.16 0.15 from
(ref.17).
-+ i _+ + +
0.05 0.02 0.02 0.03 0.03
0.76 0.63 0.33 0.Ii 0.ii
+ + -+ -+ +
02 c o n s u m p t i o n
0.06 0.05 0.02 0.02 0.02
372 was evident on 02 c o n s u m p t i o n stimulated either by FCCP or ADP
(ref. 17),
s u g g e s t i n g a direct inhibition of electron flow in the r e s p i r a t o r y chain by Cr(VI) rather than of energy transfer. Cr(III) on 02 c o n s u m p t i o n
When the effects of Cr(VI) "in situ"
mitochondria
as CrCI 3 did not have any effect
(ref. 17). and Cr(III)
were d i r e c t l y tested on thymocyte
(digitonin p e r m e a b i l i z e d thymocytes),
it was evident
that the d o s e - d e p e n d e n t i n h i b i t i o n of the maximal 02 c o n s u m p t i o n rate by c h r o m i u m d e p e n d e d on the o x i d i z a b l e substrate used succinate,
a flavin adenine d i n u c l e o t i d e
100-~
(A)
(Fig. 2,
(A) and
(FAD)-linked substrate,
100
80.
80
60,
60
(B)). With
and in the
(B)
o
o
o 40
40-
20-
20-
120 250 500 K2Cr20 7 (HM)
1~)00
120 250
500 CrCI 3 (~M)
lPoo
Fig. 2. E f f e c t s of K 2 C r 2 0 7 and CrC13 on maximal 02 c o n s u m p t i o n rate of digitonin treated rat thymocytes. 50x106 cells/ml were treated with 2-i,000 ~M K 2 C r 2 0 7 (A) and w i t h 120-1,000 ~M CrCI 3 (B) in BSS for 10 m_in. After 5 min at 30oc, 50 HM d i g i t o n i n and 5 uM cytochrome C were added. The m e d i u m was supplemented w i t h 2 m M glutamate and 2 m M malate (e-e) or with i0 m M succinate and 2 pM rotenone (o-o). FCCP was 0.4 uM and was p r e c e d e d by the a d d i t i o n of 1 ~g oligomycin/ml. Modified from (ref.17).
p r e s e n c e of rotenone w h i c h causes the c o m p l e x i to be totally reduced,
50% inhi-
bition o f FCCP stimulated 02 c o n s u m p t i o n was o b t a i n e d w i t h 400 ~M Cr(VI) and 200 ~M Cr(III); while u s i n g glutamate p l u s malate, n i c o t i n a m i n e a d e n i n e dinucleotide
(NAD+)-linked substrates,
50% inhibition was o b t a i n e d with 20 uM
Cr(VI) and 400 pM Cr(III). Q u a l i t a t i v e l y similar results were o b t a i n e d in isolated rat liver m i t o c h o n d r i a
(ref.17).
These findings indicate that the inhibition of the mitochondrial r e s p i r a t i o n by Cr(VI)
is more severe when N A D - l i n k e d substrates are oxidized,
concentrations
373 o f Cr(VI)
in the low m i c r o m o l a r range b e i n g active. These results are in good
a g r e e m e n t w i t h those r e p o r t e d by Ryberg and Alexander
(ref.28)
for the inhibi-
tory action of chromate on 02 c o n s u m p t i o n in isolated liver mitochondria. The molecular m e c h a n i s m of Cr(VI)
and Cr(III)
p h o s p h o r y l a t i o n remains to be established:
inhibition of oxidative
Cr(VI) m i g h t accept electrons de-
r i v i n g m a i n l y from N A D - l i n k e d substrates at the level of c o m p l e x e l e c t r o n t r a n s p o r t chain,
1 of the
thus i n h i b i t i n g o x y g e n r e d u c t i o n and/or o x i d i z e
essential components of the r e s p i r a t o r y chain; Cr(III), d e r i v e d from Cr(VI) reduction, m i g h t form c o o r d i n a t i o n complexes w i t h such components involved in e l e c t r o n transport. Since NADH m i t o c h o n d r i a l pool r e p r e s e n t s the p r i m a r y e n d o g e n o u s electron source in the cell,
the inhibition of m i t o c h o n d r i a l r e s p i r a t i o n by c h r o m i u m
may be e x p e c t e d to have great c o n s e q u e n c e s
Glutathione
in c y t o p l a s m i c Cr(VI)
for the cell physiology.
reduction
Given that a key step in the toxic action of Cr(VI) r e d u c t i o n to Cr(III),
is its intracellular
the v a r i a t i o n s of g l u t a t h i o n e pool c o m p o s i t i o n induced in
rat thymocytes by treatment with d i c h r o m a t e have been d e t e r m i n e d by means of HPLC a n a l y s i s
(ref. 19). Glutathione
(GSH) is indeed the main cytosolic r e d u c t a n t
and it has been r e p o r t e d to n o n - e n z y m a t i c a l l y reduce Cr(VI) s i g n i f i c a n t rate under conditions of p h y s i o l o g i c a l pH
to Cr(III) at a
(ref. 13).
Rat thymocytes have been found to c o n t a i n 2.2 - 4.6 m M GSH and 53 - 100 ~M glutathione disulphide cells
(GSSG), a s s u m i n g an intracellular volume of 0. i ~i/i06
(ref.29).
Table 5 shows that treatments of thymocytes with d i c h r o m a t e induced a strong
TABLE 5 V a r i a t i o n s of glutathione pool c o m p o s i t i o n i n d u c e d in rat thymycytes by K 2 C r 2 0 7 The cells were treated w i t h 1.0 - 2.0 m M K 2 C r 2 0 7 in BSS for 60 and 120 min. The c o n c e n t r a t i o n s of g l u t a t h i o n e pool c o m p o n e n t s are e x p r e s s e d as n m o l e s / m g of cell protein. Time of treatment (min) 60
120
Additions of K 2 C r 2 0 7 (mM)
GSH
None 1.0 2.0 None 2.0
3.2 2.8 2.4 7.4 4.2
aGlut = glutamate bAspart = aspartate
GSSG
GSH/GSSG
aGlut
bAspart
Glut + Aspart
21.3 10.8 8.0 53.0 27.7
32.6 30.2 29.4 87.6 76.1
(nmol/mg prot) 0.09 0.14 0.22 0.13 0.41
35 20 ii 57 I0
11.3 19.4 21.4 34.6 48.4
374 decrease in the ratio of r e d u c e d / o x i d i z e d glutathione, d e p e n d e n t on the dose and d u r a t i o n of exposure,
suggesting an involvement of GSH in the intracellular
reduction p r o c e s s of Cr(VI)
to Cr(III)
in these cells. Furthermore,
a marked
increase in the intracellular glutamate levels accompanied by a p a r a l l e l decrease in aspartate levels was o b s e r v e d
(Table 5). The intracellular accumula-
tion of glutamate could be due to the inhibition of its m i t o c h o n d r i a l c a t a b o l i s m by Cr(VI), which decreases the a v a i l a b i l i t y of the o x i d i z e d nicotinamide adenine dinucleotides by i n t e r a c t i n g at the level of c o m p l e x i of the electron transport chain.
CONCLUSIONS A summary scheme of major events involved in the p r o d u c t i o n of cytotoxic effects b y c h r o m i u m is p r e s e n t e d in Fig.
co% o%ds-- c§IxeE2DO0 ' --OS--o%C! -- -ODI Cr (Vl) compounds~
H
~1 -MEDIATED .... TRANSPORTH L;ru4
~
3.
-.)I-0/_~..~/,CD
:r. . . . ' bru4
/2crC~llic)omplePxe ~ ~ DNA-'PROTEIN U~72_ ~i I~R~ ~ L'rU4 ~ ,-; ~'!1 n
..
ADPJ
Fig. 3. S u m m a r y scheme of major events involved in c h r o m i u m toxic action at c e l l u l a r level. M o d i f i e d from: A.G. Levis and V. Bianchi, in S. Langard (Ed.), Biological and Environmental Aspects of Chromium, Elsevier, N o r t h - H o l l a n d Biomedical Press, Amsterdam, 1982, p.171.
375 A l t h o u g h Cr(VI)
has been p r o v e d to p r o d u c e membrane
m o d i f y i n g m e m b r a n e - l i n k e d proteins
(e.g. Mg2+-ATPase),
functional damage by its cytotoxic action
seems to be m a i n l y c o n f i n e d at intracellular level. Given that the transport of Cr(III) Cr(VI)
complexes p o s s i b l y by endocytosis is e x t r e m l y limited, e x t r a c e l l u l a r r e d u c t i o n by a variety o f systems,
including serum proteins,
acid and c o m p o n e n t s in the outer surface of the cell membrane, i m p o r t a n t m e c h a n i s m of c h r o m i u m detoxification. Therefore,
ascorbic
represents an
the a b i l i t y o f Cr(VI)
to produce cytotoxic e f f e c t s is strictly related to its facilitated transport across the p l a s m a m e m b r a n e and its reduction to Cr(III) sibly t h r o u g h the intermediate reactive Cr(V) w i t h i n the c y t o p l a s m Cr(VI) cysteine,
is reduced in : (I) cytosol,
ascorbate and D T - d i a p h o r a s e
NAD(P) H - d e p e n d e n t flavoprotein for the process;
inside the cell, pos-
form.
(ref. 15) -
(2) microsomes,
P 4 5 0 - r e d u c t a s e e n z y m a t i c system;
where GSH but also
- the latter b e i n g a F A D - c o n t a i n i n g are a m o n g the m o l e c u l e s responsible
mainly by the NAD(P) H - d e p e n d e n t cytochrome (3) mitochondria,
where Cr(VI)
m i g h t accept
e l e c t r o n s d e r i v i n g m a i n l y from N A D - l i n k e d s u b s t r a t e s at the level of c o m p l e x 1 of the e l e c t r o n transport chain;
(4) nuclei.
I n t r a c e l l u l a r c h r o m i u m has been shown to interfere with specific steps of c e l l u l a r energy m e t a b o l i s m in that it p r o d u c e s severe unbalance of purine ribonucleotide p o o l s and inhibition of m i t o c h o n d r i a l o x y g e n consumption. However,
it is not yet k n o w n to w h a t extent the effects caused by Cr(VI)
are d i r e c t l y related to the r e d u c t i o n p r o c e s s or to the subsequent binding of Cr(III)
and p o s s i b l y o f Cr(V)
to biological molecules w h i c h p l a y a critical
role in cell physiology.
ACKNOWLEDGEMENTS This work was supported b y grants from the N a t i o n a l R e s e a r c h Council of Italy
(CNR, P r o g e t t o F i n a l i z z a t o Medicina P r e v e n t i v a e Riabilitativa,
p r o g e t t o Rischio Tossicologico,
C R 83.02707.56)
and the V e n e t i a Region
Sotto(Centro
Regionale di Alta S p e c i a l i z z a z i o n e in C a n c e r o g e n e s i Ambientale). We a c k n o w l e d g e P. Arslan,
M. Beltrame and T. Pozzan for m a k i n g a v a i l a b l e
u n p u b l i s h e d o b s e r v a t i o n s on EPR.
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