Chem.-Biol. Interactions, 47 (1983) 363--371 Elsevier Scientific Publishers Ireland Ltd.
363
IN VIVO INTERACTIONS OF A C R Y L O N I T R I L E WITH MACROMOLECULES IN RATS
MOHAMMED Y.H. FAROOQUI and AHMED E. AHMED*
Department of Pathology and Department of Pharmacology and Toxicology, The University of Texas Medical Branch at Galveston, Galveston, TX 77550 (U.S.A.) (Received December 29th, 1982) (Revision received June 8th, 1983) (Accepted June 9th, 1983)
SUMMARY
The irreversible binding of [2,3-'4Chacrylonitrile ( V C N ) t o proteins, RNA and DNA of various tissues of male Sprague--Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LDs0) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. R N A and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.
Key words: Acrylonitrile -- Macromolecules -- DNA alkylation -- Irreversible binding INTRODUCTION
Acrylonitrile (CH2=CH--CN, VCN) m o n o m e r is a well known neurotoxicant with a wide range of commercial applications including the preparation of polymeric materials necessary for the production of synthetic fibers, *To whom correspondence should be sent. Abbreviations: BSA, bovine serum albumin; CBI, covalent binding index; CIP, chloroform/isoamyl alcohol/phenol (24 : 1 : 25) ; CNS, central nervous system; HAP, hydroxyapatite; NaP, sodium phosphate; UNaP-SDS-EDTA, 8 M urea/0.24 M sodium phosphate/ 1% sodium dodecyl sulphate/10 mM EDTA; VCN, acrylonitrile. 0009-2797/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
364 synthetic rubber, plastics and in some countries for grain fumigation. In 1981 VCN ranked 39th among organic c o m p o u n d s synthesized in the United States [1 ]. Previous research identified acute VCN toxicity in rats including adrenocorticolysis [2,3], tissue damage of brain, liver, lung and kidney [4], central nervous system (CNS) dysfunction and congestive lung edema [5] and rapid depletion of reduced glutathione [6--8]. The chronic VCN toxicity includes adrenal atrophy and insufficiency [9], papillomas of stomach, zymbal gland carcinomas, tumors of CNS and masses of mammary region [10]. Studies of the biotransformation of VCN point to an epoxidation of the vinylic double bond by mixed function oxidase system [11]. Both the parent molecule and the epoxide are electrophilic and reactive towards glutathione and other nucleophilic sites of tissue macromolecules [11--17 ]. In view of the multipotent nature of VCN as a carcinogen, it was considered of interest to investigate the irreversible binding of VCN in vivo to nucleic acids and proteins from target tissues of rats. MATERIALS AND METHODS Chemicals [2,3-~4C]Acrylonitrile (2.8 mCi/mmol, 99%+) was obtained fom Pathfinder Laboratories Inc., St. Louis, MO. Radiochemical purity was determined by gas chromatography using a Perkin Elmer Model 3920 chromatQgraph, equipped with FID and 6' × 1/8" stainless steel column packed with chromosorb 102 (80--100 mesh} at 130°C with N2 used as a carrier gas. The specific activity of the dosing solution was adjusted through dilution with unlabeled acrylonitrile (Aldrich Chemical Co., Milwaukee, WI}. All other chemicals were reagent grade. Animals and treatment Male Sprague--Dawley rats (200--250 g) from Charles River, Wilmington, MA, were acclimatized in our animal facility, with free access to food and water. Each rat from each group (3--4 per group} received a single oral dose of 46.5 mg VCN kg -1 (50 p C i . kg -1) dissolved in 0.5 ml distilled water. This dose represents 0.5 LDs0 for rats [15]. Animals were killed at 1, 6, 24 and 48 h after administration of VCN. Organs including liver, kidney, brain, spleen and stomach were dissected out and frozen rapidly on solid CO2. The pooled organs from each group of rats were stored at - 7 0 ° C until isolation of macromolecules. Protein isolation from tissues Proteins were isolated according to the m e t h o d of Beland et al. [ 1 8 ] , with few modifications. Frozen tissues were minced and homogenized in 10 vol. of 8 M urea/0.24 M sodium phosphate/l% sodium dodecyl sulfate/ 10 mM EDTA (UNaP-SDS-EDTA) (pH 6.8) with a m o t o r driven teflon Potter-Elvehjem homogenizer, cooled with ice for 5 min and homogenized
365 again. The homogenizing cooling sequence was repeated five times, and the homogeneous solution was shaken to insure complete lysis. An equal volume of chloroform/isoamyl alcohol/phenol ( 2 4 : 1 : 2 5 , CIP) saturated with UNaP-SDS-EDTA, was then added and mixed thoroughly for 15 min. The emulsion resulting from CIP extraction was separated into two phases by centrifugation at 4000 rev./min for 15 min in an IEC Model K centrifuge equipped with a No. 838 swinging bucket head. The CIP phase was removed and saved for protein isolation while UNaP-SDS-EDTA layer was extracted at least one additional time with CIP. Following CIP partitioning the aqueous phase was treated twice with diethyl ether to remove trace amounts of phenol. This consisted of adding an equal volume of diethyl ether, mixing thoroughly and centrifuging as above. At this point the UNaP-SDS-EDTA phase which contained the nucleic acids was stored at 4°C for up to 72 h or applied directly to the hydroxyapatite (HAP) column. Proteins were precipitated from the combined CIP extracts by the addition of equal volume of acetone. The solution was mixed for 15 min and the precipitate was isolated by centrifugation. The proteins residues were resuspended in acetone and again isolated by centrifugation. This process was repeated with diethyl ether and ethanol and the washed protein residues were then dried in a vacuum desiccator.
R N A and DNA isolation by HAP chromatography The aqueous extract from the CIP partitioning was applied to an HAP column [18]. The column media was prepared by suspending 1 g DNA-grade hydroxyapatite (Bio-Rad Labs., Richmond, CA) per mg of DNA in 0.014 M sodium phosphate (NAP) (pH 6.8), by gently swirling the slurry and by decanting the fines. This process was repeated with UNaP and the suspension was poured into a 2.5 X 40 cm glass column. A peristaltic pump was used to pump one column volume of UNaP at a flow rate of approx. 1 ml/min. The aqueous nucleic acid solution was then applied and the elution pattern was monitored by LKB 2089 UVICORD III UV detector (254 nm). UNaP was passed through the column until the absorbance returned to zero. This was followed by 0.014 M NaP (pH 6.8) to purge the urea from the system. After re-establishing the initial absorbance, 0.48 M NaP (pH 6.8) was applied to the column to elute DNA. The UNaP, which contained RNA, and the 0.48 M NaP, which contained DNA, were dialyzed against 5 mM Bis-Tris, 0.1 mM EDTA (pH 7.1) and then concentrated by either lyophilization or evaporating in vacuo on a rotary evaporation at 38°C. In some instances the I~NA and RNA were precipitated by making solutions 0.1 M in NaCI followed by the addition of two volumes of cold ethanol. The concentrations of proteins, RNA and DNA were determined respectively by the m e t h o d of Bradford [19] with bovine serum albumin (BSA) as the standard, by the orcinol reaction [20] with yeast RNA (type XI, Sigma) as the standard and by the m e t h o d of Burton [20] with calf t h y m u s DNA as the standard. Radioactivity was determined by liquid scintillation counting after addition of 15 ml of Aquasol® (New
366 England Nuclear, Boston, MA) in Searle (Tracor) Scintillation Counter (Searle Analytic Inc., Des Plaines, IL). RESULTS
Irreversible protein binding in vivo Figure 1 illustrates the time dependence of the irreversible protein binding of [2,3-14C]VCN in various tissues of rats. Initially in the first hour after oral administration of VCN the highest protein binding occurred in spleen and stomach followed by liver, brain and kidney. After 6 h of treatment the protein binding plateaued until 48 h. HAP chromatography of RNA and DNA As shown in Fig. 2 the separation of RNA and DNA fractions was accomplished by applying the aqueous phases resulted from the phenol extraction of proteins from the tissue homogenates of liver (A), stomach (B) and brain (C) to HAP column. The RNA fraction eluted with low ionic strength buffer of 0.24 M phosphate (UNaP) (pH 6.8) in the fractions beginning from 30 to 80 ml whereas the DNA fraction eluted with the high ionic strength buffer of 0.48 M NaP (pH 6.8) between the fractions 190 and 230 ml. The amounts of radioactivity recovered in the eluted RNA and DNA fractions is shown in Fig. 2.
Registration of radioactivity in RNA Radioactivity from [2,3-14C]VCN could be registered in RNA from all the organs studied (Fig. 3). RNA from liver displayed the highest amounts of radioactivity followed by brain and stomach. The liver RNA showed
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Fig. 1. Radioactivity in proteins (calculated as nmol equivalents of VCN b o u n d / r a g protein) of various rat tissues at different time intervals after a single oral dose of 46.5 mg/kg b o d y wt. - , liver; A, s t o m a c h ; - , brain; X, kidney; *, spleen. Each value represents mean +- S.D. of 3 animals.
367
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FRACTIONS(ml) Fig. 2. HAP c h r o m a t o g r a m s o f D N A and R N A f r o m liver (A), s t o m a c h (B) and brain (C) of rats t r e a t e d w i t h [2,3~4C ]-VCN. , absorbance; • 8, radioactivity.
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Fig. 3. Radioactivity in R N A (calculated as nmol V C N bound/mg R N A ) of rat liver (=), brain (A), and stomach (*) at different time intervals after a single oral dose of 46.5 rag/ kg body wt. Each value is mean + S.D. of 3 determinations.
368 150: cI: z Q to bz w
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Fig. 4. Radioactivity in D N A (calculated as p m o l VCN b o u n d / m g D N A ) of rat liver (m), s t o m a c h (*) and brain ( . ) at different time intervals after a single oral dose o f 46.5 m g / kg b o d y wt. Each value is m e a n *_ S.D. o f 3 determinations.
maximal levels of radioactivity at 6 h after administration whereas RNA from other organs showed maximal levels at 24 h. The radioactivity in R N A of all the three organs remained unchanged after 24 h until 48 h.
Registration of radioactivity in DNA As depicted in Fig. 4, significant amounts of radioactivity from [2,314C] VCN could be registered in brain D N A followed by gastric DNA. D N A from brain displayed the highest amounts of radioactivity followed by that of stomach and liver. The D N A from all the three organs showed maximal levels of radioactivity at 24 h which remained unchanged thereafter. The promising quantitative correlation of D N A binding in vivo with the carcinogenic response is generally expressed as covalent binding index (CBI) [22]. The CBI have frequently been used to make comparisons of different compounds eventually studied in different laboratories and under TABLE I C O V A L E N T B I N D I N G I N D I C E S F O R V C N IN V A R I O U S T I S S U E S O F R A T S A n i m a l s were sacrificed 24 h after oral administration of 46.5 m g V C N (50 u C i ) / k g b o d y wt. Tissue
ttmol V C N b o u n d a per mol D N A
CBI b
Liver Stomach Brain
5.1 -+ 0.6 45.1 _+ 6.8 56.4 +- 7.9
5.9 51.9 65.3
aValues are m e a n s +- S.D. o f 3 determinations. b C B I are calculated as the ratio o f u m o l V C N b o u n d / t o o l D N A t o m m o l V C N applied/kg b o d y wt. [ 2 2 ] .
369 various experimental conditions. This is expressed as damage to DNA/dose. The molar units for such expressions allow a very rapid visualization of h o w many molecules are bound per mole of DNA. Table I shows the quantitative data on binding of VCN to DNA nucleotides. Estimation of CBI for the target organs studied reveal the maximal CBI for brain reaching up to 65.3 at 24 h and remained unchanged thereafter, followed by tha¢ for stomach and the lowest CBI are for the liver. DISCUSSION Experimental animals dosed with 14C-labeled VCN exhale 14CO: and H14CN in addition to unmetabolized VCN [13,15,16]. Despite its remarkable volatility only a b o u t 5% of the total dose of VCN administered was exhaled unchanged [16]. The available metabolic data suggest that the highly reactive electrophilic molecule of VCN is metabolized by more than one pathway. One of these, the direct alkylation (cyanoethylation) of the cellular nucleophile glutathione (GSH) results in its depletion and consequently the excretion of appropriate mercapturic acids [8]. The in vitro irreversible protein binding [ 2 3 ] , the in vivo covalent binding to erythrocyte [12] and other tissue proteins [15,16], all suggest that similar interaction might be involved with respect to sulfhydryl groups of other nucleophilic sites in the macromolecules. The radioactivity registered in R N A and DNA of various rat tissues after the administration of [2,3-'4C] VCN in this study indicates the irreversible binding of VCN and/or its reactive metabolites. The presence of radioactivity and estimation of CBI (Table I) in brain nucleic acids both suggest a role of VCN in initiation of brain tumors, which has been shown in studies of occupational VCN exposure [24]. Extensive interactions with gastric macromolecules may indicate a possible role in development of tumors [24], ulcers and VCN-induced gastrointestinal bleeding demonstrated by Ghanayem et al. [25]. It must be pointed out here that the observations made in this study show DNA alkylation by VCN after a single oral dose. However, the carcinogenic response has been observed after chronic exposure [ 2 4 ] . . Guengerich et al. [17] have found that in vitro binding of 2-cyanoethylene oxide, a metabolite of VCN, to nucleic acids was more extensive than was the binding of VCN itself. The nature of these adducts is not yet known, and a variety of products was formed when 2-cyanoethylene oxide was incubated with ribonucleosides [17]. One of the adducts formed under such conditions was 1,N6-ethenoadenosine and a mechanism for formation of such adduct can be predicted on the basis of other studies [26]. Such reaction has been found to be a major step in the alkylation of nucleic acids by metabolites of vinyl halides [27]. Preliminary studies (data not shown) in our laboratory using high performance liquid chromatography of ISNA hydrolysates showed elution of radioactivity in the peaks corresponding to standard DNA nucleosides deoxyadenosine and deoxyguanosine. The role
370 o f such p r o d u c t s f o r m e d b y either d e o x y a d e n o s i n e or d e o x y g u a n o s i n e remains u n c e r t a i n until such a p r o d u c t can be isolated f r o m D N A in vivo. In a d d i t i o n to d e p l e t i o n o f tissue GSH, VCN is k n o w n t o r e a c t none n z y m a t i c a l l y w i t h t h e c y s t e i n e t h i o l groups o f p r o t e i n s [ 6 - - 8 , 1 7 ] . Appare n t l y t h e G S H c o n j u g a t i o n plays a m o r e i m p o r t a n t role in VCN m e t a b o l i s m [ 1 7 ] , t h a n does e p o x i d e h y d r o l a s e in d e t o x i c a t i o n o f a n y 2 - c y a n o e t h y l e n e o x i d e g e n e r a t e d in vivo [ 1 7 ] . GSH-transferase and e p o x i d e h y d r o l a s e b o t h m a y have some p r o t e c t i v e role in d e t o x i c a t i o n o f VCN. Regarding the possible relationship o f such i n t e r a c t i o n s o f VCN to the t u m o r i g e n i c responses s h o w n in studies o f o c c u p a t i o n a l VCN e x p o s u r e [24], the d i r e c t m i c r o s o m a l e p o x i d a t i o n o f VCN b y brain tissues [11] or possible migration of e p o x i d e f o r m e d in the liver to target tissues, s t o m a c h and brain [ 1 7 ] , c o u l d a c c o u n t for the a l k y l a t i o n o f R N A and DNA. Since t h e r e are k n o w n tissue variations in rates of D N A repair, it is conceivable t h a t t h e D N A damage in brain tissue m a y have a longer half-life t h a n t h a t in liver w h e r e it is rapidly repaired. This c o u l d result in t h e so far d e m o n s t r a t e d t u m o r i g e n i c responses o n l y in e x t r a h e p a t i c tissues. T h e isolation o f a d d u c t s f o r m e d b y a l k y l a t i o n b y VCN and studies o n D N A damage and repair should be c o n s i d e r e d to u n d e r s t a n d the m e c h a n i s m o f VCN i n t e r a c t i o n s with macromolecules. ACKNOWLEDGEMENTS These studies were s u p p o r t e d b y G r a n t E S 0 1 8 7 1 f r o m National Institute of Health. The skillful assistance o f Mrs. R u t h B u f f i n g t o n in preparing this m a n u s c r i p t is gratefully a c k n o w l e d g e d . REFERENCES 1 P.L. Layman, Big volume chemicals' output fell again in 1981, Chem. Eng. News, 3 May (1982) 11. 2 S. Szabo and H. Seyle, Adrenal apoplexy and necrosis produced by acrylonitrile, Endokrinologie, 57 (1971) 405. 3 S. Szabo, E.S. Reynolds and K. Kovacs, Acrylonitrile induced adrenal apoplexy, Am. J. Pathol., 82 (1976) 653. 4 R. Schultka, R. Schmidt, G. Wagner and E. Franzen, Morphologische unter suchungen zur wirkung von acrylonitrile in subcronischen intoxications versuch bei der weiben ratte, Gegenbaurs Morphol. Jahr., 121 (1975} 609. 5 G. Paulet and J. Denson, L'acrylonitrile toxicite -- Mechanisme -- D'Action Therapeutique, Arch. Int. Pharmacodyn. Therap., 131 (1961) 54. 6 S. Szabo, K.A. Bailey, P.J. Boor and R.J. Jaeger, Acrylonitrile and tissue glutathione: differential effect of acute and chronic interactions, Biochem. Biophys. Res. Commun., 79 (1977) 32. 7 M.Y.H. Farooqui and A.E. Ahmed, Effect of acrylonitrile and potassium cyanide on red cell metabolism, Fed. Proc., 40 (1981) 678. 8 B.I. Ghanayem and A.E. Ahmed, In vivo biotransformation and biliary excretion of [1-14C ]acrylonitrile in rats, Arch. Toxicol., 50 (1982) 175. 9 S. Szabo, E.S. Reynolds, P. Komanicky, M.T. Moslen and J.C. Melby, Effect of chronic acrylonitrile ingestion on rat adrenal, Toxicoh Appl. Pharmacoh, 37 (1976) 133.
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