141

Chem.-BioL Interactions, 75 (1990) 141--151 Elsevier Scientific Publishers Ireland Ltd.

M E C H A N I S M S O F C H R O M I U M T O X I C I T Y IN M I T O C H O N D R I A

DAVID RYBERG ~* and J A N ALEXANDER b

"Institute of Occupational Health, P.O. Box 8149, Dep. 0033 Oslo 1 and bNational Institute of Public Health, DepL of Toxicology, Geitmyrsveien 75, 0462 Oslo 4 {Norway) (Received October 4th, 1989) (Revision received January 25th, 1990) (Accepted February 14th, 1990)

SUMMARY

The oxygen consumption of isolated rat heart mitochondria was potently depressed in presence of 1 0 - 5 0 p_M Na2CrO 4 when NAD-linked substrates were oxidized. The succinate stimulated respiration and the oxidation of exogeneous NADH in sonicated mitochondria were not affected by chromate at this concentration range. A rapid and persistent drop (40% in 2 rain) in the mitochondrial NADH level was observed after chromate addition (30 pM) under conditions which generally should promote regeneration of NADH. Experiments with bis-(2ethyl-2-hydroxybutyrato)oxochromate(V) and vanadyl induced reduction of Cr(VI) in presence of excess NADH were performed. These experiments indicated that NADH may be directly oxidized by Cr(V) at physiological pH. The activity of 10 different enzymes were measured after lysis of intact mitochondria pretreated with chromate ( 1 - 1 0 0 / ~ I ) . Na2CrO 4 at a very low level (3--5 ~M ) was sufficient for 50% inhibition of a-ketoglutarate dehydrogenase. Higher concentrations (20--70 pM) was necessary for similar effect on ~-hydroxybutyrate and pyruvate dehydrogenase. The other enzymes tested were unaffected. Thus, the chromate toxicity in mitochondria may be due to NADH depletion as a result of direct oxidation by Cr(V) as well as reduced formation of NADH due to specific enzyme inhibition.

Key words: Chromium - Mitochondria -- Respiratory inhibition mechanisms

* To whom all correspondence should be addressed. 0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

Toxic

142 INTRODUCTION Health hazards associated with exposure to hexavalent chromium (Cr(VI)) compounds have been known for decades. A wide spectrum of effects are described, varying from skin ulcers and allergic dermatitis [1] to serious kidney injury [2] and increased risk of cancer in the respiratory tract [3]. The molecular mechanisms of the cytotoxic as well as the genotoxic effects are only partially understood. However, it is generally assumed that these are associated with the oxidizing power of Cr(VI) and with the ability of trivalent chromium (Cr(III)) to form substitution-inert complexes with a number of cell components. Water soluble Cr(VI)-compounds, in contrast to Cr(III), easily penetrate the cell membrane. Within the cell Cr(VI) is reduced to Cr(III) which is bound in organic complexes. Although the reductive metabolism is suggested to play a key role in the mechanism of Cr(VI) toxicity, relatively few compounds have been identified which reduce Cr(VI) at physiological pH. Among several low molecular weight compounds studied only ascorbate and thiols reduce Cr(VI) at significant rates in vitro [4]. Certain enzymes in the cytosol [5,6], endoplasmic reticulum [7,8] and mitochondria [9] of mammalian cells have also been reported to stimulate Cr(VI)-reduction (in presence of electron donor substrates). In a previous study with rat liver mitochondria results were presented which indicated that Cr(VI)-reduction was closely coupled to the powerful and selective inhibition of NADH-linked respiration [9]. In the present study we further examined the mechanisms of this inhibition. Rat heart mitochondria were chosen since they have a high resiratory capacity and potentially an increased reduction state in the NAD-pool. MATERIALS AND METHODS Rotenone, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), aketoglutarate dehydrogenase (bovine heart), nicotinamide adenine dinucleotide oxidized (NAD) and reduced (NADH) form, nicotinamide adenine dinucleotide-phosphate (NADP and NADPH) and all other biochemicals were products from Sigma Chemical Company. Nagarse (from B. subtilis) was supplied from Serva, [1-"C]pyruvate was purchased from Amersham. All other chemicals were commercially available and of p.a. quality. Wistar rats (300--400 g) from Mollegaard, Copenhagen, were used in all experiments. The animals were starved 12 h before sacrifice by decapitation.

Preparation of mitochondria Hearts from 4--6 rats were rapidly chilled in 150 mM KC1 and 1 mM EGTA at 0 °C and finely minced with scissors. To each gram of tissue 10 ml of H-medium (150 mM KCI, 20 mM K-HEPES (pH 7.25) and 2 mM EGTA) with 2 mg Nagarse was added and incubated at 0 °C for 10 min. The minced tissue was washed once in medium without Nagarse and suspended in Hmedium with 1 mM ATP and 0.5% BSA. The homogenizaion was per-

143 formed by six strokes with loose pestle and three strokes with tight pestle in a Dounce homogenizer. The 1:10 diluted homogenate was then centrifuged at 700 x g (max) for 10 rain in about half-filled tubes (Sorvall SS-34). The resulting supernatant was centrifuged at 10 000 x g (max) for 8 min. The mitochondrial pellet was resuspended in H-medium and centrifuged at 10 000 x g (max) for 8 min. The last step was repeated once, and the final pellet was resuspended in 250 mM sucrose, 20 mM K-HEPES (pH 7.20) to a total protein concentration of 50--80 mg/ml (Biuret). Rat liver mitochondria were prepared as described previously [9].

O~-measurement The oxygen consumption was measured with a Clark 02 electrode as described [9] in a medium containing 150 mM KC1, 20 mM K-HEPES (pH 7.20), 5 mM K-phosphate, 0.5 mM MgC12 and 0.3 mM EDTA. This medium was used in all mitochondrial incubations.

CrlVII and NADH measurements Cr(VI) was analyzed by a modification of the diphenylcarbazide (DPC) method as published by Ryberg [10]. NADH was measures as described by Sahlin [11].

Synthesis of Crf~-complex The synthesis of bis-(2-ethyl-2-hydroxybutyrato)oxochromate(V) was performed as described by Krumpolc et al [12].

Enzymatic measurements All enzymatic activities except the pyruvate dehydrogenase measurement were performed in a Schimadzu MPS 2000 spectrophotometer equipped with thermostated cuvettes. The activities of malate dehydrogenase (EC 1.1.1.37), isocitrate dehydrogenase (NAD) (EC 1.1.1.41), isocitrate dehydrogenase (NADP) (EC 1.1.1.42), a-ketoglutarate dehydrogenase complex, ~-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and glutamate dehydrogenase (EC 1.4.1.3) were determined by the appearance/disappearance of NAD(P)H at 340 nm in KCNtreated mitochondria [13]. Ketoacyl-CoA thiolase (EC 2.3.1.9) was measured by following the breakdown of acetoacetyl-CoA at 303 nm [14]. The activity of citrate synthase (EC 4.1.3.7) was assayed by the dithiobis-(2-nitrobenzoate) (DTNB) method [13] and succinate cytochrome c reductase as described by Woitczak and Zaluska [15]. Pyruvate dehydrogenase was determined as 14C02 liberation after 10 rain incubation at 37 °C with [1-14C]pyruvate [16]. RESULTS The oxygen consumption in a suspension of rat heart mitochondria was significantly depressed in presence of sodium chromate in the low micromolar range. This inhibition was only seen when the mitochondria oxidized

144 % of control (a)

t

100 90

•~ ,

•-hydroxybutyrate

80

• ~ •

citrate

70

•

* pyruvate

÷

+ NADH, sonicated mitochondria

o

o succinate

70

8"0

60 50 40

2O tO 1"0

~ oo

20

30

40

5"0

60

90

IO0~uM Na2CrO 4

(b)

\ 02 50 juM N a 2 C r O 4

1

I

'

ml

kcontrol

25pM

Na 2 C r O 4

Fig. 1. (a) Effect of Na2Cr04 on the mitochondrial respiration with different substrates. The results are the mean of three experiments. Rat heart mitochondria (0.5 mg/ml protein) were preincubated for 4 min at 37°C in 2 ml buffer (see Materials and Methods) with 1 mM malate and 10 mM of the indicated substrates. In experiments with succinate 5 pM rotenone replaced malate. Na2CrO 4 was then added and the mixture transferred to a thermostated chamber equipped with a Clark 02~lectrode. After 3 more min the respiration was released with 0.5 ~M FCCP. Experiments with sonicated mitochondria were performed as described above with mitochondria oxidizing pyruvate. The incubation mixture was sonicated 3 min after chromate addition. NADH (1 raM) was added before the O£measurements. The specific respiratory rates expressed as nmol 02 x rain -~ x mg protein -1 were 438, 42, 165, 145 and 475 with pyruvate,/3hydroxybutyrate, succinate, citrate and NADH respectively. The O£consumption during the initial 2 rain after FCCP addition was used as basis for calculation. (b) Mitochondrial oxygen consumption after 4 rain preincubation with chromate. Typical traces are shown. The experiment was performed as described in the legends to (a) with 10 mM pyruvate added.

145 NAD-linked s u b s t r a t e s (Fig. la). T h e r e s p i r a t i o n of m i t o c h o n d r i a i n c u b a t e d s e v e r a l m i n u t e s with 50 ~M Na2CrO 4 was n e a r l y c o m p l e t e l y blocked w h e n p y r u v a t e w a s oxidized (Fig. lb). To e v a l u a t e w h e t h e r t h e electron t r a n s p o r t chain was affected, t h e 02-consumption was m e a s u r e d with sonicated mitochondria in t h e p r e s e n c e of c h r o m a t e and with e x t e r n a l l y added N A D H as t h e p r i m a r y electron source. No inhibition w a s s e e n e v e n w h e n this was conf i r m e d d u r i n g p y r u v a t e s t i m u l a t e d r e s p i r a t i o n in intact m i t o c h o n d r i a before sonication (Fig. la). This o b s e r v a t i o n and the exclusive s e n s i t i v i t y of NADlinked r e s p i r a t i o n indicate t h a t t h e e l e c t r o n t r a n s p o r t chain was not directly involved in t h e m e c h a n i s m s of c h r o m a t e inhibition. Depletion of i n t r a m i t o c h o n d r i a l N A D H or inhibition of its g e n e r a t i o n s e e m e d m o r e likely as inhibitory m e c h a n i s m s . E x p e r i m e n t s w e r e t h e r e f o r e p e r f o r m e d to m e a s u r e possible changes in t h e N A D redox-level a f t e r addition of c h r o m a t e . A significant d r o p (40%) in mitochondrial N A D H was found 2 rain a f t e r addition of 30/~M Na2CrO 4 (Table I). T h e d e c r e a s e in N A D H persisted for at l e a s t 5 rain e v e n if the m i t o c h o n d r i a w e r e incubated in p r e s e n c e of r o t e n o n e and excess of s u b s t r a t e . (The changes in N A D could not be measu r e d in p r e s e n c e of c h r o m a t e due to t h e r e a c t i v i t y of Cr(VI) d u r i n g the acid N A D - e x t r a c t i o n procedure.) T h e s e r e s u l t s indicate t h a t Cr(VI) m a y affect both t h e f o r m a t i o n and oxidation of N A D H . A direct oxidation of N A D H b y Cr(VI) s e e m s unlikely since b o t h a r e quite stable w h e n m i x e d t o g e t h e r at p H a b o u t 7 [4]. H o w e v e r , m o r e r e a c t i v e c h r o m i u m species m a y be involved in a direct oxidation of mitochondrial N A D H . T w o d i f f e r e n t e x p e r i m e n t a l p r o c e d u r e s w e r e p e r f o r m e d to e v a l u a t e w h e t h e r N A D H m a y be d i r e c t l y oxidized b y p e n t a v a l e n t c h r o m i u m (Cr(V)).

TABLE I REDUCTION OF Cr (VI) AND OXIDATION OF NADH IN RAT HEART MITOCHONDRIA The figures are the mean with S.D. from three experiments. Rat heart mitochondria (1.0 mg/ml protein) were preincubated for 4 rain at 37°C with 10 mM pyruvate and 5 ~M rotenone in a total volume of 8.0 ml buffer (see Materials and Methods). Three aliquots of 1 ml, each in duplicate, were withdrawn from the incubate. The first sample (0 min) was taken just before addition of 3 0 ~M NasCrOd (final) and the second and third 2 and 5 min after Cr-addition, respectively. In the control experiments no chromate was added. Cr(VI) was measured (in separate experiments) with DPC after alkalinization and extraction of interfering materials as described in Materials and Methods. In the '0 min' sample Na~CrO~was added after alkalinization (30 pM final). NADH was extracted with etanolic KOH and analyzed by a bioluminescence method as described in Materials and Methods. Incubation time after Cr-addition (rain)

Exposed mitochondria

Control

Cr (VI) (nmol/ml)

NADH (nmol/ml)

NADH (nmol/ml)

0

30.0 '

6.51 ± 0 . 2 3

6.45 ± 0.30

2

27.3 _

0.86

3.58 ± 0.20

--

5

2 4 . 4 __ 0.91

2.86 ± 0.25

6 . 5 2 ± 0.23

146 First, freshly synthesized bis-(2-ethyl-2-hydroxybutyrato)oxochromate(V) was added in equimolar quantity to a buffered solution of NADH. The reaction was completed within 2 - - 3 s using 0.1 mM NADH and Cr(V)~complex in 100 mM K-HEPES buffer (pH 7.20) and 22°C (recorded at 340 nm, data not shown). This was too fast for a reliable registration with the applied equipment. In the second experimental procedure Cr(VI)-reduction was performed with the fast one-electron donor vanadyl cation (VO 2÷) as reducing agent, either used alone or in combination with NADH. As expected three equivalents of vanadyl were necessary for complete reduction of Cr(VI) to Cr(III). However, in presence of 10 times molar exess of NADH (relative to chromate), only one equivalent of :anadyl-ion was sufficient (Fig. 2). This experiment indicates the following reactions: Cr(VI) + 3V(VI)-* Cr(III) + 3V(V)

(1)

Cr(VI) + V(IV)-~ Cr(V) + V(V) Cr(V) + N A D H - * Cr(III) + N A D

(2)

Remaining Cr(Vl)

"I

lO0

90 8O 70 60 5O

~X X

40 30 20

mM NADH

10

\

i> | 2 3 equivalents V 0 2 + added Fig. 2. Reduction of Cr(VI) d u r i n g stepwise addition of VO2÷. Effect of NADH. The result is the m e a n of two experiments. One ml of 100 ~M Na2CrO ~ in distilled water or in mixture with 1 m M NADH was incubated at room temperature under N2-flushing. An anaerobe solution of 10 m M VOSO, in 10 m M citrate buffer (pH 6.0) was added slowly during constant stirring. After addition of the indicated quantity of vanadyl, the sample was diluted four times with 50 m M N a H C O 3 pH 6.8 and Cr(VI) m e a s u r e d as described in Materials and Methods. The DPC reagent was added immediately a n d OD a t 540 n m m e a s u r e d a f t e r 10 rain. I n t e r f e r e n c e d u e to V02 ÷ was corrected for in parallel experiments where V02C1 replaced VOSO 4. |

1

147 In e x p e r i m e n t s described in this paper, c h r o m a t e addition caused a rapid and p e r s i s t e n t drop in mitochondrial N A D H level even when the conditions generally should p r o m o t e r e g e n e r a t i o n of N A D H . To evaluate w h e t h e r chromate also affected the formation of N A D H , we m e a s u r e d the activity of several N A D H - p r o d u c i n g e n z y m e s after lysis of mitochondria p r e t r e a t e d with chromate. Since l a r g e r quantities of mitochondria w e r e used in this s t u d y and c h r o m a t e has similar effects on the respiration in h e a r t and liver mitochondria, r a t liver mitochondria w e r e used. Of 10 e n z y m e s t e s t e d only ]3hydroxybutyrate dehydrogenase, a-ketoglutarate dehydrogenase and p y r u v a t e d e h y d r o g e n a s e w e r e affected when the concentration of Na2CrO 4 was less than 100 ~Vl (Table II). a - K e t o g l u t a r a t e d e h y d r o g e n a s e was, however, v e r y sensitive to c h r o m a t e as only 3 - - 5 pM was required for 50% inhibition. Addition of r e d u c e d dithiols did not r e a c t i v a t e the enzyme. The purified a - k e t o g l u t a r a t e d e h y d r o g e n a s e (from bovine heart) was also inhibited by Na2CrO 4 although less sensitive t h a n the mitochondrial enzyme. The inhibition increased with time and was most p r o m i n e n t when the e n z y m e was p r e i n c u b a t e d with a - k e t o g l u t a r a t e and c h r o m a t e (Fig. 3a). This inhibition was also seen when N A D was s u b s t i t u t e d by ferricyanide as elect r o n acceptor (Fig. 3b).

TABLE II INHIBITION OF ENZYMES IN MITOCHONDRIAINCUBATED WITH Na2CrO4 Rat liver mitochondria were preincubated in 2 ml 150 mM KC1, 20 mM K-HEPES (pH 7.20), 5 mM phosphate buffer and 1 mM KCN for 4 min at 37°C. For citrate synthase and thiolase measurements 5 mM glutamate was added to the preincubation mixtures. For all other enzyme measurements the actual substrate was present. Na2CrO~ (0-100/~M) was then added and lysis preformed by 0.1°/0 Lubrol PX or sonication (for ~-hydroxybutyrate DH). After dividing the mixture equally in sample and reference cuvette, the essential factors and cosubstrates were added to the sample cuvette only (see Materials and Methods). Pyruvate dehydrogenase was measured by ~4C02liberation after addition of [1-~4C]pyruvateas described in Materials and Methods. The correction for endogenous NAD(P)H oxidation was performed in separate experiments by addition of known quantities of NAD(P)H. Mitochondrial enzyme

Na2CrO4(pM) to attain 500/0 inhibitiona

Citrate synthase Isocitrate dehydrogenase, NAD Isocitrate dehydrogenase, NADP ~-Ketoglutarate dehydrogenase Succinate dehydrogenase Malate dehydrogenase Pyruvate dehydrogenase Glutamate dehydrogenase /3-Hydroxybutyrate dehydrogenase Ketoacyl-CoAthiolase

n.i°

n.i. n.i. 3--5 n.i. n.i. 60 -- 70 n.i. 25--30 n.i.

•n.i., No inhibition with Na2CrO, less than 100 pM. The figures are the results of six experiments.

1

2

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f

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3

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4

5

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0.01

.02

.03

.04

.05

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2O

1

2

,,,"

3

.,,"

/

°°°~° °,."

4

5

°°°.°'°" °.°°° °°°'°

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6

min. incubation

Fig. 3. (a) Reduction of NAD by purified ~-ketoglutarate dehydrogenase. Effect of chromate. Purified a-ketoglutarate dehydrogenase complex (from bovine heart) was diluted in 100 mM Na-phosphate {pH 7.2) with 0.5 mM MgCl~ and 10 mM dithiothreitol. The complete reaction mixture contained 100 mM Na-phosphate (pH 7.2), 3 mM ~-ketoglutarate (KG), 15 ~1 enzyme mixture (to 1 ml cuvette), 0.5 mM NAD and 0.2 mM CoA. In experiments with chromate, 20 ~M Na~CrO 4 (final) was added 1 rain before the reaction was started either by NAD or wketoglutarate addition. The reduction of NAD was performed at room temperature and recorded at 340 nm ....... control; - - , 20 ~M Na2Cr04, reaction initiated by KG; ---, 20 ~ NazCrO 4, reaction initiated by NAD. (b) Reduction of ferricyanide by purified a-ketoglutarate dehydrogenase. Effect of chromate. The procedure was as described in legend to (a), but 0.5 mM K-ferricyanide replaced NAD. The incubation mixture also contained 0.2 mM thiaminopyrophosphate and 0.5 ml~ MgC12. The reduction of ferricyanide was recorded at 420 nm ..... control; ,20 pM NazCr04, reaction initiated by KG.

0.1

.2

.3

.4

.5

O D , ~40

oo

149

DISCUSSION The inhibitory effect of Na2CrO 4 on NAD-linked respiration seen in rat heart mitochondria was nearly identical to that previously described for rat liver mitochondria [9]. In both cases chromate at a concentration range 1 500 ~M did not affect the succinate stimulated respiration. Neither was any inhition seen in sonicated mitochondria oxidizing added NADH, although the inhibition of NAD-linked respiration was seen before sonication. These experiments probably exclude the electron transport chain as the site for Crinhibition. Under conditions which generally should promote generation and preservation of NADH, there was a significant drop in the mitochondrial NADH reduction state after chromate addition. Since NADH is quite stable in presence of Cr(VI) at physiological pH , the observed oxidation of NADH may involve more reactive species of chromium. Formation of pentavalet chromium (Cr(V)} is described in Cr(VI) exposed microsomes [17] and mitochondria [18] and also in mixtures of Cr(VI) and reduced thiols or complex organic mixtures [19,20]. The stable Cr(V) compound bis-(2-hydroxy-2-methylbutyrato)oxochromate(V) has previously been used as oxidizing agent during neutral pH conditions [21,22]. When this compound is mixed with NADH in buffered solution the reaction is immediate. NADH is therefore directly oxidized by Cr(V). A similar conclusion can be drawn from experiments with induced reaction between Cr(VI) and NADH using VO2÷ as a one electron inducer. In this experiment Cr(V) is probably formed from Cr(VI) and one electron from the oxidation of the vanadyl ion [23]. Although NADH may serve as electron source for Cr(VI) reduction, only few electrons are consumed in this process compared with the high capacity for NAD-reduction in heart mitochondria. Thus, a reduced regeneration of NADH is probably the most important explanation for the drop in NADHlevel seen in Cr(VI)-treated mitochondria. The activity of several different enzymes were therefore measured after lysis of chromate treated mitochondria. With Na2CrO 4 in the concentration range up to 100 pM only 3 of 10 enzymes tested were affected, i.e., a-ketoglutarate dehydrogenase (KGDH) pyruvate dehydrogenase (PDH) and ~-hydroxybutyrate dehydrogenase (HBDH). The inhibition of these 3 enzymes may explain the observed respiratory inhibition in liver mitochondria incubated with chromate and the selected substrates. The effect of chromate on enzymes in heart mitochondria was not measured, but a similar mechanism for respiratory inhibition seems probable. The potent Cr(VI)-inhibition of mitochondrial KGDH may be parallel to that obtained with arsenite and cadmium (Cd2÷). These two last agents work through their binding to the dithiol group in the lipoyl moiety [24]. The lipoyl moiety may also be involved in the chromate inhibition of KGDH (and PDH), as indicated in experiments with ferricyanide as electron acceptor for the dehydrogenase reaction. Inhibition of chromate treated glutathione reduc-

150 tase and xanthine oxidase is probably due to Cr(III) binding at essential thiol groups [25,26]. Although thiols are excellent ligands for stable Cr(III) complexes [27,28] this alone can not explain the selective inhibitory effect of chromate. The existence of electron donors for Cr(VI)-reduction in the vicinity of potential Cr(III~ligands may be more important for this selectivity. Whether KGDH is capable of reducing Cr(VI) is not known, however, our results may indicate this. The mechanism of inhibition of mitochondrial pyruvate dehydrogenase may be similar to that of KGDH since these two enzymes have similar properties [29]. f~-Hydroxybutyrate dehydrogenase (HBDH) is localized in the inner mitochondrial membrane and its activity is dependent on this environment [30]. The vulnerability of HBDH against chromate may be linked to this locality or to its lipophilic parts since soluble enzymes catalyzing similar reactions i.e. malate dehydrogenase and lactate dehydrogenase [31] were not affected by chromate. This study demonstrate that the toxicity of Cr(VI) at low doses is expressed in a rather specific way in isolated mitochondria. Only few enzymes are known to be affected by chromium in the low micromolar range [31]. Of these KGDH in rat liver mitochondria is the most sensitive. Depending on the cellular and mitochondrial uptake, a depressed energy metabolism is expected in exposed cells and tissues. Such effects has been observed in isolated liver cells [9] and thymocytes [32]. REFERENCES 1

N.B. Pedersen, in: S. Langaard (Ed.), Biological and Environmental Aspects of Chromium, Elsevier, Amsterdam, 1982, pp. 249-275. 2 S.K. Tandon, in: S. Langaard (Ed.), Biological and Environmental Aspects of Chromium, Elsevier, Amsterdam, 1982, pp. 209-220. 3 S. Langaard and T. Norseth, in: L. Friberg, G.F. Nordberg and V.B. Vouk (Eds.), Handbook on the Toxicology of Metals, Vol. 2, Elsevier Science Publishers, 1986, pp. 185--210. 4 P.H. Connett and K.E. Wetterhahn, Metabolism of the carcinogen chromate by cellular constituents, Struct. Bonding, 54 (1983) 9 3 - 1 2 4 . 5 R.B. Banks and R.T. Cooke, Chromate reduction by rabbit liver aldehyde oxidase, Biochem. Biophys. Res. Commun., 137 (1986) 8 - 1 4 . 6 S. De Flora, A. Morelli, C. Basso, M. Romano, D. Serra and A. De Flora, Prominent Role of DT-diaphorase as a cellular mechanism reducing chromium(VI) and reverting its mutagenicity, Cancer Res., 45 (1985) 3188-3196. 7 J.D. Garcia and K.W. J e n n e t t e , Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate, J. Inorg. Biochem., 14 (1981) 281-295. 8 A. Mikalsen, J. Alexander and D. Ryberg, Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450, Chem.-Biol. Interact., 69 (1989) 175-192. 9 D. Ryberg and J. Alexander, Inhibitory action of hexavalent chromium (Cr(VI)) on the mitochondrial respiration and a possible coupling to the reduction of Cr(VI), Biochem. Pharmacol., 33 (1984) 2 4 6 1 - 2466. 10 D. Ryberg, Problems with methods for measurement of hexavalent chromium applied to biological samples, Acta Pharmacol. Toxicol., Suppl. 7, 59 (1986) 624--626.

151 11 12

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17 18 19

20

21 22 23 24 25 26 27

28

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