Oxidation of inactive trivalent chromium to the mutagenic hexavalent form

167

Research, 58 (1978) 167--173 © Elsevmr/North-Holland Bmmedmal Press

Mutation

OXIDATION OF INACTIVE TRIVALENT CHROMIUM TO THE MUTAGENIC HEXAVALENT FORM

FERNANDO L. PETRILLI and SILVIO DE F L O R A

Institute o f Hyglene, Unwerszty o f Genoa, Via Pastore 1, 16132 Genoa (Italy) (Received 13 January 1978) (Revision received 17 April 1978)

(Accepted 26 April 1978)

Summary Soluble trivalent chromium compounds (chrommm potassium sulfate, chrom m m nitrate, chromium chloride, n e o c h r o m m m and chromium alum) were inactive for Salmonella typhlmurium TA100, even at mflhgram amounts per plate. No effect could be detected either m the absence or in the presence of rat-hver, lung or muscle mmrosomal fractions, of rat-muscle mltochondria (with or w i t h o u t ATP), of oxidized glutathione (GSSG), or of human serum, plasma or erythrocyte lysates. Conversely, addihon of a strongly oxidizing agent (potassmm permanganate) resulted in toxic effects in plates incorporating more than 40--80 pg of compounds and elicited a dose-effect mutagenic response at 10--40 pg per plate. These effects could be ascribed to oxidation o f chromium from the trivalent to the active hexavalent state. Insoluble chromite, as tested m the spot test, was spontaneously mutagenic, owing to contamination of the industrial product with hexavalent chromium. The results obtained may be useful to mterpret the findings of carcmogeniclty tests and to predict health hazards hnked to chrommm.

Introduction

In previous investigations [5,16,17] we have demonstrated that trivalent chromium is mactive for his- strains of Salmonella typh~murium (TA1535, TA1537, TA1538, TA98 and TA100), whereas the hexavalent form is toxin or mutagenm, depending on concentration of the metal tested with bacteria. In particular, the results obtained suggested t h a t the hexavalent ion interacts with Salmonella DNA and induces, at sub-lethal doses, both frameshift errors Abbrewatzons

thlone.

ATP, adenosme-5'-tnphosphate, GSH, reduced glutathlone, GSSG, oxldlzed gluta-

168 and base-pair substitutions. The mutagenic response is enhanced through an error-prone recombmation repair of the damaged nucleic acid. However, the mutagenic effects disappeared m the presence either of rat-liver mmrosomal fractions or of human erythrocyte lysates. Such metabohc deactivation could be ascribed to reductmn of hexavalent chromium to the inactive tnvalent form. Reducing metabohtes, chiefly TPNH, which accumulate m the cell as a consequence of enzymic oxidative pathways, appeared to be responsible for the above effect. Interestingly, mmrosomal fractmns from rat lung, even at the same protein concentration as with the liver preparations, were very poorly active in reducing the mutagenic potentml of hexavalent chromium. Ratmuscle preparations, human serum and plasma were also completely machve. These results correlate quite well with the epidemmlogical evidence supporting an elective localization of chrommm-induced cancer m human lung [2,3,4,10, 12], which is the only organ where chrommm shows a progressive accumulation during life [20]. Moreover, these laboratory fmdmgs might account for the controversml results of annnal carcmogeniclty tests, which have generally demonstrated the development of tumors in rodents, but only at implant sites [9,11,15,18,19]. Ewdence that trlvalent chromium c o m p o u n d s can also yield subcutaneous sarcomas m rats, although with a lower frequency and with a longer lncubatmn period as compared with hexavalent chrommm [13,14], prompted us to check whether mactwe tnvalent chromium, in the form of varmus compounds, can be converted into the mutagenic hexavalent state when incubated with So typhimurium in the presence of an omdizing agent or of metabohc systems. Materials and methods

Chemicals Trivalent chrommm was tested as CrK(SO4)2 • 12H20 (chromium potassmm sulfate) and CrC13- 6H20 (chromium chloride) (B.D.H., Poole, England), as Cr(NO3)3" 9H20 (chrommm mtrate) (Rledel De Haen, Hannover, West-Germany), and in the form of three industrial products, i.e. chromite, chromium alum and neochrommm, kindly supphed by Dr. Cesare Maltonl (Istltuto dl Oncologla "Felice Addarli", Bologna, Italy) and which were the same products as used in the previously cited carclnogenlclty tests. The percentage composlh o n of the three products used, as reported by the producers (Montedison, Spinetta Marengo (Alessandria), Italy) was the followmg: chromite = Cr203 44--46, Fe203 29--30, A1203 15--16, $102 0.5--3, CaO 0.5--2; chromium alum = Cr2(SO4)3 37--39, K2SO4 16--18, H20 41--43; n e o c h r o m m m = Cr(OH)SO4 56--58, Na2SO4 23--24. KMnO4 (potassium permanganate) (Merck Co., Darmstadt, West-Germany) was used as oxidizing agent, and ascorblc acid (Noury--Baker N.V., Deventer, Holland) as reducing agent. Adenoslne-5'-trlphosphate (ATP) and oxidized glutathlone (GSSG) were commercially available (Boehrmger Bmchemma, Mannhelm, West-Germany). With the exceptmn of chromite, whmh is Insoluble, and of ATP, whmh was dissolved m Trls buffer (pH 7.6), all c o m p o u n d s were dissolved in distilled

169 water, at the concentrahons mdmated under Results, and sterihzed by filtration through HAWPO2500 membranes (Millipore S.A., Buc, France).

Bacterial strata TA100 was used as bacterial tester strain, because it had been found to be the most sensitive hts- S. typhzmurmm strain m reveahng c h r o m m m mutageniclty [16,17]. S. typh~murmm strams were kindly supphed by Dr. Bruce N. Ames (Department of Bmchemistry, Umversity of Californm, Berkeley, Cahf., U.S.A.). Mutagemc~ty and chemzcal assays Soluble c h r o m m m compounds were tested by the plate-incorporation test, and msoluble compounds by the spot test, according to the methods described by Ames et al. [1]. Mutageniclty assays were comparahvely performed m the absence and in the presence of reducmg or oxidizing chemicals or of metabohc systems (see Results). Human erythrocyte lysates and the S-9 mix contmning microsomal preparatmns (S-9 fractions) from rat-liver, lung or muscle homogenates (9000 × g supernatants), hawng protein concentratmns of 37, 15.5 and 8 mg/ml, respectively, were prepared as previously described [17]. Supernatants (4000 × g ) o f rat-muscle homogenates, having a protein concentration of 12.4 mg/ml, were used as source of m~tochondrm. The presence of hexavalent chromium was checked by using the s-diphenylcarbamde (Carlo Erba, Milan, Italy) reagent [6]. Results

The results of the plate-mcorporahon test (Table 1) showed that each of the soluble trivalent c h r o m m m compounds under scrutiny was, per se, inactive for S. typhzmurmm TA100, even at milligram amounts per plate. Conversely, the addition of KMnO4 to top agar (100 pg per plate) resulted in toxin effects in plates incorporating more than 40--80 pg of tnvalent chromium compounds and ehcited a dose-effect mutagemc response at 10--40 pg per plate. These effects could be ascribed to oxidation of the metal from the tnvalent to the hexavalent state, as revealed by production of a reddish-purple color after the a d d l h o n of the s-dipheny]carbazlde reagent. As shown m Table 2, the number of h~ + revertants induced by trivalent c h r o m m m compounds was n o t slgmfmantly changed m the presence of rathver, lung or muscle mmrosomal fractions, or of rat-muscle mltochondria (either with or w i t h o u t addltmn of ATP). S~mlarly, no effect was apparent on the addlhon of h u m a n serum, plasma or erythrocyte lysates to the soft agar overlay. GSSG was also ineffective. On the other hand, the results of the spot test showed that the chromite used was spontaneously mutagemc for S typh~murium (Fig. 1). However, such activity could be ascribed to the presence of hexavalent chromium in the mdustrlal product under test. In fact, mutagenlclty was reduced in the presence of ascorbm acid, and the product spontaneously reacted with dlphenylcar-

170 TABLE 1 N U M B E R O F T A 1 0 0 R E V E R T A N T S Y I E L D E D BY S O L U B L E T R I V A L E N T C H R O M I U M COMP O U N D S I N T H E A B S E N C E A N D I N T H E P R E S E N C E O F K M n O 4 ( 1 0 0 ~g P E R P L A T E ) Compound

Controls

Amount per p l a t e (#g)

0

h~ + revertants per plate KMnO~

KMnO~

1 9 4 -+ 24

2 0 7 -+ 31

CrK(SO4) 2 • 12H20 ( c h r o r m u m p o t a s s m m sulfate)

8000 80 40 20 10

175 187 208 195 202

TEa TEa 880 525 385

Cr(NO3) 3 9H20 (chrommm mtrate)

8000 80 40 20 10

181 194 177 212 198

TEa TE a TEa 880 515

CrCI 3 6 H 2 0 (chrormum chloride)

8000 80 40 20 10

172 203 191 215 202

TEa TEa TEa 985 490

Cr(OH)SO 4 Na2SO4 (neochromlum)

8000 80 40 20 10

179 192 207 198 210

TEa TEa 740 495 320

Cr2(SO4) 3 K2SO 4 -H20 (chromium alum)

8000 80 40 20 10

170 192 209 185 212

TEa TEa 930 545 410

a T E = toxin e f f e c t s ( a b s e n t o r s p a r e b a c k g r o u n d l a w n ) .

TABLE 2 N U M B E R O F T A 1 0 0 R E V E R T A N T S Y I E L D E D BY C r K ( S O 4 ) 2 1 2 H 2 0 IN T H E A B S E N C E A N D IN T H E P R E S E N C E O F P R E P A R A T I O N S F R O M R A T T I S S U E S O F OR H U M A N B L O O D C O M P O N E N T S Metabohc system

None L i v e r S-9 m i x a L u n g S-9 m i x a Muscle S-9 m t x a Muscle m l t o c h o n d n a b Muscle m l t o c h o n d r l a + A T P (2 ~ m o l e s ) Muscle m l t o c h o n d n a + A T P ( 1 0 ~ m o l e s ) Human serum b Human plasma b H u m a n e r y t h r o c y t e lysate b

+ hzs r e v e r t a n t s p e r p l a t e Control

CrK(SO4) 2 12H20 (1 m g p e r p l a t e )

1 8 9 +- 27 178 184 201 235 195 192 194 181 167

176 -+ 22 167 180 195 214 187 178 185 170 158

a 5 0 ~1 m l c r o s o m a l f r a c t i o n m 0 5 m l S-9 m i x p e r p l a t e b 0.1 m l p e r p l a t e

171

Fig. 1. S p o t test, p e r f o r m e d b y p l a c i n g 2 m g o f c h r o m t t e , c o n t a m i n a t e d w i t h h e x a v a l e n t c h r o m m m , a t the c e n t e r o f t h e s o f t agar o v e r l a y e m b e d d i n g S t y p h w n u r m m T A 1 0 0

bazide. Addition of KMnO4 to top agar enhanced the mutagenm effect of chromite. Discussion The results of the present study complete the findings of previous inveshgations on chromium mutagemclty and metabolism in vitro [5,16,17], by reproducmg m the Salmonella test system the interconversion processes that occur between the mactive tnvalent form and the active (toxic or mutagenic) hexavalent form of this metal. In particular, reduction from hexavalent to trivalent chromium, leading to deactivation of mutagemcity of the metal, was readily obtained both in the presence of reducing agents, such as ascorbm acid, sodmm sulfite, reduced glutathione (GSH), DPNH and TPNH, and of metabolic systems, such as hver microsomal fractions and erythrocyte lysates [17]. This mdmates that reduchve processes are prevalent m these biologmal systems. Conversely, the shift of tnvalent to hexavalent chromium could be demonstrated m the present study only after the addition of a strongly oxidizing

172 agent. Such conversion could not be mimicked in vitro by metabolic systems, n o t even by those, such as lung or muscle microsomal preparations, that failed to deactivate hexavalent c h r o m m m mutagenicity. No metabolic activation of trlvalent chromtum was afforded by rat-muscle mltochondria, supplemented by ATP, or by oxidized glutathlone (GSSG). Similarly, the addition of serum or plasma had no effect. With reference to this, it is n o t e w o r t h y that trivalent chromium entering the blood stream is strongly b o u n d to plasma proteins, whereas hexavalent chromium is selectively concentrated in erythrocytes [7], where It undergoes metabohc deactivation to the trivalent form [ 17 ]. Therefore, at variance with the reverse process, the metabolic activation of c h r o m m m to the mutagemc form does n o t appear to be reproducible by metabohc systems m a short-term assay. Provided that tnvalent c h r o m m m is actually oxidized to the hexavalent form in the o r g a m s m - as postulated by Grogan [8] - - t h i s should eventually be a slow process occurrmg only under partmular conditions of retention and accumulation of the metal, and only in hssues with a low reducing potenttal. In this view, it is interesting to remember that carcinogenmlty tests had been performed by Maltom et al. [13,14] by using the same three industrial products tested m this study. All c o m p o u n d s had been injected subcutaneously (1 ml of sahne containmg 30 mg of each c o m p o u n d ) into rats (40 antmals per compound). Neochromium yielded sarcomas at the site of injection m 10 animals (25%) and chrommm alum m 8 ammals (20%), with mean latency permds of 78 and 72 weeks, respechvely. These values, though slgmficant, are considerably lower than those recorded in the same set of experiments by testing hexavalent chrommm compounds. Thus, the frequency of sarcomas m rats mjected with hexavalent chrommm pigments (chromium yellow, chrom m m orange and m o l y b d e n u m orange) was 65 to 90%, the latency permd being 32 to 40 weeks only. A slow conversmn from the machve trlvalent form to the mutagenm and carcmogenlc hexavalent state m sltu might well account for these patterns. Surpmsmgly, the same chromite that we found to be mutagemc for S typhim u n u m , even without artffmml oxidation, owing to traces of hexavalent chromium, failed to mduce tumors in any of the 40 anmlals tested by Maltoni et al. The presence of traces of hexavalent chromium in chromite might represent an ~mpumty of the mdustrml product used, or alternatively, result from ox~datmn of Cr203. Among the varmus components of the product, the high amounts (29--30%) of Fe203 might have been responsible for such parhal oxldatmn. Anyhow, the possibility that a tnvalent chrommm p r o d u c t m a y be contaminated wtth the achve hexavalent form should be taken into account m predmtlng occupatmnal hazards hnked to this metal.

Acknowledgements We thank Drs. C. Benmcelh and P. Zanacchi for their excellent assistance.

References 1 Ames, B N , J McCann and E Yamasakl, Methods for detecting carcmogens and mutagens Salmonella/mamrnahan m l c r o s o m e m u t a g e n l c l t y t e s t , M u t a h o n R e s , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4

with the

173 2 BaetJer, A M., P u l m o n a r y c a r c i n o m a m c h r o m a t e w o r k e r s I A r e v i e w o f t h e h t e r a t u r e a n d r e p o r t of cases, A r c h I n d . H y g . , 2 ( 1 9 5 0 ) 4 8 7 - - 5 0 4 3 B l d s t r u p , P . L , a n d R . A . M . Case, C a r c i n o m a o f t h e lung m w o r k m e n m t h e b l c h r o m a t e - p r o d u c m g i n d u s t r y m G r e a t Britain, Br J. I n d M e d , 13 ( 1 9 5 6 ) 2 6 0 - - 2 6 4 . 4 B r i n t o n , H.P., E.S. Frasler a n d A L K o v e n , M o r b i d i t y a n d m o r t a h t y e x p e r i e n c e a m o n g c h r o m a t e w o r k e r s , P u b l H e a l t h R e p . , 67 ( 1 9 5 2 ) 8 3 5 - - 6 4 7 . 5 De F l o r a , S , M e t a b o h e d e a c t i v a t i o n o f m u t a g e n s m t h e S a l m o n e l l a / m m r o s o m e t e s t , N a t u r e ( L o n d o n ) , 271 ( 1 9 7 8 ) 4 5 5 - - 4 5 6 . 6 Ege Jr., J F., a n d L S l l v e r m a n , S t a b l e c o l o r l m e t n c r e a g e n t for c h r o m m m , A n a l C h e m , 19 ( 1 9 4 7 ) 693 7 G r a y , S.J., a n d K S t e r h n g , T a g g i n g of r e d cells a n d p l a s m a p r o t e i n s w i t h r a d l o a c h v e e h r o m m m , J C h n I n v e s t , 29 ( 1 9 5 0 ) 1 6 0 4 - - 1 6 1 3 8 G r o g a n , C.H , E x p e r i m e n t a l studles m m e t a l c a n c e r o g e n e s t s V I I I O n the e t i o l o g i c a l f a c t o r m c h r o m a t e c a n c e r , C a n c e r , 10 ( 1 9 5 7 ) 6 2 5 - - 6 3 8 9 H u e p e r , W . C , a n d W W. P a y n e , E x p e r t m c n t a l s t u d i e s m c a n c e r o g e n e m s . C h r o m i u m , m c k e l , t r o n a n d arsemc, Arch Environ. Health, 5 (1962) 445--462 10 I . A R C., C h r o m i u m a n d m o r g a m c c h r o m m m c o m p o u n d s , m M o n o g r a p h s o n t h e E v a l u a t i o n of C a r c i n o g e n i c Risk o f C h e m m a l s t o Man, I n t e r n a t i o n a l A g e n c y f o r R e s e a r c h on Cancer, L y o n , 1 9 7 3 , pp 100--125 11 L a s k m , S , M K u s c h n e r a n d R . T D r e w , Studies m p u l m o n a r y c a r c m o g e n e s m , m : M . G H a n n a J r , P N e t t e s h e t m a n d J R. G t l b e r t ( E d s ) , I n h a l a h o n C a r c i n o g e n e s i s , U S. A t o m i c E n e r g y C o m m i s s i o n S y m p o s t u r e Series N o 18, 1 9 7 0 , p p . 3 2 1 - - 3 5 1 12 M a l t o m , C., C Slnlbaldl a n d P C h m c o , S u b c u t a n e o u s s a r c o m a s m rats f o l l o w i n g l o c a l r e j e c t i o n of c h r o m m m orange and m o l y b d e n u m orange, Excerpta Medlca Int. Congr S e t , 322 (1974) 19--26 13 M a t t o n l , C , G L e f e m m e , P. Chleco a n d D C a r r e t h , La e a n c e r o g e n e s l a m b l e n t a l e e p r o f e s s l o n a l e n u o v e p r o s p e t h v e alia luce della c a n c a r o g e n e s l da clorttro dl wnfle, Osp V l t a , 1 ( 1 9 7 4 ) 4 - - 6 6 1 4 M a l t o n l , C., P r e d l c h v e c a r c m o g e n l c l t y b l o a s s a y s m m d u s t x m l o n c o g e n e m s , m p r e p a r a t i o n . 15 N e t t e s h e l m , P , a n d A S H a m m o n s , I n d u c h o n o f s q u a m o u s cell c a r c i n o m a m the r e s p i r a t o r y t r a c t of m i c e , J Natl. C a n c e r I n s t , 47 ( 1 9 7 1 ) 6 9 7 - - 7 0 1 16 Petrtlll, F L , a n d S De F l o r a , T o x i c i t y a n d m u t a g e m c l t y o f h e x a v a l e n t c h r o m l t t m o n Salmonella typh~murturn, A p p l E n w r o n . M l c r o b l o l , 33 ( 1 9 7 7 ) 8 0 5 - - 8 0 9 17 Petrllh, F L , a n d S De F l o r a , M e t a b o h c d e a c h v a h o n of h e x a v a l e n t c h r o m i u m m u t a g e m c l t y , M u t a t i o n Res., 54 ( 1 9 7 8 ) 1 3 9 - - 1 4 7 18 R o e , F J.C , a n d R L C a r t e r , C h r o m m m c a r c i n o g e n e s i s c a l c m m c h r o m a t e as a p o t e n t c a x c m o g e n for t h e s u b c u t a n e o u s h s s u e s of t h e r a t , Br. J C a n c e r , 23 ( 1 9 6 9 ) 1 7 2 - - 1 7 6 19 S a n d e r m a n J r , F.W., M e t a l c a r c i n o g e n e s i s m e x p e r i m e n t a l anLrnals, F o o d C o s m e t . T o x l c o l , 9 ( 1 9 7 1 ) 105--120 2 0 S c h r o e d e r , H A , T h e role o f c h r o m m m in m a m m a h a n n u t r i t i o n , A m J C h n N u t r , 21 ( 1 9 6 8 ) 2 3 0 - 244