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The effect of aflatoxin B1, aflatoxin B2 and sterigmatocystin on nuclear deoxyribonucleases from rat and mouse livers
Chem.-Biol. Interactlons
353
Elsevier Pubhshmg Company, Amsterdam Printed m The Netherlands
T H E E F F E C T OF A F L A T O X I N B1, A F L A T O X I N B2 A N D S T E R I G M A T O CYSTIN ON NUCLEAR DEOXYRIBONUCLEASES FROM RAT AND M O U S E LIVERS
M J PITOUT a, H A M c G E E a AND J C SCHABORT u
aDlvtston of Toxicology, National Institute for Nutritional Diseases, S A Medical Research Council, Pretorta, and t, Department of Biochemistry, Rand AJmkaans University, Johannesburg (Repubhc of South Africa) (Recewed December 4th, 1970) (Accepted January 29th, 1971)
SUMMARY
Certain carcinogens have an effect on the activity of pancreatic deoxynbonuclease I (DNAase I, EC 3 1 4 5) The effect of two potent mycocarclnogens, vtz. aflatoxln B1 and stengmatocystm, as well as the weak carcinogen, aflatoxm B2, on the activity of two nuclear DNAases (DNAases I and II) from rat hver was therefore investigated. No significant interaction between aflatoxm B1 and the two enzymes was observed. Stengmatocystm ts almost insoluble in aqueous solutions and therefore could not be used for m v i t r o studies. I n v t v o experiments, however, clearly indicated a marked effect of aflatoxln BI on the total activity of DNAase II. F r o m these results it is suggested that aflatoxm B1 could be a precarcmogen and that it may be converted, probably by mlcrosomal enzymes, to the active carcinogenic compound. Alternatively, aflatoxm B1 could react with the natural inhibitor, resulting in higher enzyme activity In the normal cell, activity of the enzyme must be under rigorous control in order top reserve the integrity of the genetic message Abnormal circumstances, causing release of activity, might result in anomalies in the genetic expression mechanism. The negative result obtained with aflatoxln B2 illustrates the fact that the double-bond of the terminal furane ring plays a vital role in determining the toxic and carcinogenic activity of aflatoxm B 1
Stengmatocystin produced no effect on either enzyme, suggesting that the mechanisms of carcinogenic action of the two toxins are different. The fact that orally dosed aflatoxln B1 has no effect on the activity of DNAase II from mouse liver nuclei could support the concept that the mouse is capable of convertmg the aflatoxln B~ to noncarcmogenlc metabohtes Abbreviation DMSO, dlmethylsulphoxlde.
Chem-Btol Interactions, 3 (1971) 353-361
354
M J. PITOUT, H A MCGEE, J. C. SCHABORT
INTRODUCTION
There have been a number of m w t r o and m v t v o studies on the effects of various potent carcinogens such as the butter yellow-type carcinogens, urethane, y-butyrolactone and dtepoxybutane on the activity of pancreatxc DNAase I. In these studies, considerable mcreases in activity were found as a result of treatment with the carcmogens 1-3. With weak carcinogens no activation of pancreatic DNAase I was observed The potent carcinogens have been found to be devoid of any effect on the actlvlty of spleen DNAase II. However, LESCAe t a l 4 demonstrated decisively a sharp rise in pulmonary DNAase II activity when rats were treated with the carcinogen benz-3,4-pyrene. It is widely, although not umversally, beheved that the basic defect in cancer is a chromosomal mutation in somatic cells. Evidence suggesting a relationship between lysosomal damage and the occurrence of chromosomal aberrations has been presented and it was, therefore, suggested that the released lysosomal DNAase might be able to penetrate the nucleus and attack the chromosomes 5,6 Although the presence of an acid DNAase (DNAase type II) in nuclei from various sources has been shown by several workers 7,8, the enzyme has never been isolated and neither has any function been ascribed to it Apart from DNAase II, alkahne DNAases (type I) which are associated with the non-hlstone fraction of chromatm have only recently been described 9-12 In the hght of the above-mentioned observations, It was decided to investigate the possible influence of the hepatocarclnogens aflatoxln B1 and sterlgmatocystm on the actlvRles of the two DNAases from rat liver. In addition, the effect of aflatoxm Be on the actlwtles of the two nuclear DNAases from rat liver was also studied Ariatoxin B2 has a slmdar molecular structure to aflatoxm B1, but it has a relatively weak toxic and carcinogenic action m rats in comparison to aflatoxln B1, the most potent carcinogen known ~3 For the sake of comparison, the effect of aflatoxm Bt and sterlgmatocystln on the activity of acid and alkaline DNAases from mouse liver nuclei was also investigated It is known that the mouse is resistant to aflatoxm B1 carcinogenesis t4. MATERIALS AND METHODS
Ammals
Random-bred male Wlstar-denved rats with an average body weight of 200 4- 10 g and male albino mice (22 :~ 2 g body weight) were used. The mice were obtained from Onderstepoort Veterinary Research Institute, Pretorm, which originally obtained them from the Wellcome Bureau of Scientific Research, London, England. Rats and mice were fed a balanced pellet diet, and were provided with tap water a d h b t t u m Rats and mice were divided into 3 groups, t.e aflatoxln B1 and stengmatocystm treated rats and the control group, respectively Each group consisted of 3 rats or 4 mice. DMSO was used as solvent for the toxins and the control animals were dosed only with DMSO Chem-Btol Interactions, 3 (1971) 353-361
EFFECT OF CARCINOGENSON MOUSEAND RAT DNAASES
355
Chemicals Thymus-gland D N A was obtamed from Sigma Chemical C o , Ohio, U.S.A All other chemicals used were of analytical reagent grade Aflatoxm B1 and stengmatocystm were isolated and purified according to STEYNis and HOLZAPFEL et al a6, respectively Aflatoxm B2 was prepared from aflatoxm B1 by means of hydrogenation according to VAN DORP et al 1 7 Extractton and tsolatton of nuclei from mouse and rat hver All operations were done in the cold (2-5 °) The nuclei were prepared according to the method of MAGGIO et al 18 with minor alterations The hvers of each group were pooled and homogenized m 0 88 M sucrose-3 m M MgC12 solution in the ratio of 10-15 g hver/100 ml sucrose solution The suspension was filtered through 4 layers of cheesecloth muslin gauze and centrifuged at 1500 × g for 10 mm The precipitate was suspended in 2 3 M sucrose-3 m M MgCI2 solution and the supernatant was recentrlfuged as described above The process was repeated twice To separate the nuclei from contaminants, the 2 3 M sucrose suspension (180 ml/10-15 g liver) was centrifuged at 44 000 < g for 65 m m The purity of the nuclei was checked by electron and light microscopy 19. Extraction o f the acid and alkahne DNAases from rat and mouse hver nuclet Since previous studies on the effect of carcinogens on DNAases were done m vttro (see refs 1-3), an attempt was made to isolate and purify these enzymes from rat liver nuclei 3 Of the two enzymes, the acid DNAase was found to be the more stable enzyme, and was, therefore, purified to a greater extent 3 For the in VlVO experiments, however, crude nuclear DNAases from rat and mouse livers were prepared by the following procedure: To lyse the nuclei, they were suspended in 5 ml of 0 05 M citrate-0 005 M mercaptoethanol buffer (pH 7.0) Centrlfugatlon at 2000 × g for 20 rain yielded a preclpitate (chromatm) and the supernatant (nuclear sap) The chromatln acidic proteins were obtained by the procedure of WANG2° The p H of the nuclear sap was carefully adjusted to 4.7 by the addition of acetic acid, and the resultant milky suspension was clarified by centnfugatlon at 10 000 × g for 20 m m to give a clear, colourless supernatant which contained the acid DNAase. Assay for acttvmes o f nuclear DNAases The activities of acid and alkahne DNAases were assayed according to HODES et al 21 and O'CONNOR 1°, respectively For both enzymes, one unit (U) of enzyme is that amount of enzyme which produces one AA26o umt/h. Specific actlvxty is expressed as unlts/mg protein Protein was determined according to the method of LOWRY et al. 2z The m vitro effect o f aflatoxm B1 on the acttvttles of actdandalkahne DNAases from rat hver In vttro investigations could not be performed with ster~gmatocystm due to its very poor solublhty A stock solution of aflatoxm B1 was made in 1,2-propylene Chem-Btol Interactions, 3 (1971) 353-361
356
M . J . PITOUT~ H A. MCGEE~ J C. SCHABORT
glycol (7 mg/ml) and different concentrations of aflatoxm B~ were prepared from the stock solution by dilution (0.2 ml aflatoxm B1 solution added to the incubation mxxture) The activities of the two nuclear DNAases in the presence of different concentratlons of aflatoxm B1 were measured as described above. Control values were obtained by using 0 2 ml of propylene glycol In vtvo effects of aflatoxm B~ and stertgmatocystm on the acttvtttes of actd and alkahne DNAases from rat hver nuclet Wlstar rats were given a dally per os dose over a 30-day period. The dady dose was eqmvalent to 1/5 LDso of aflatoxm B 1 and stengmatocystln, where the LDso values of aflatoxln B~ and sterlgmatocystln were taken as 7 mg/kg and 160 mg/kg body weight, respectively 13'23 Rats were kdled by decapitation at predetermined time intervals, the hvers excised, nuclei xsolated, the two enzymes extracted and their activities assayed as described above The effect o f aflatoxm B 2 o n the acttvtttes of the two nuclear DNAases The similarity in chemical structure between aflatoxins B1 and B2 prompted an m vtvo study of the effect of aflatoxm B2 on the actlwtles of the acid and alkaline DNAases Male Wlstar rats were dosed with aflatoxlns B~ and B2 at 1 75 mg/kg dady over a period of 18 days. Rats were kdled at predetermined time intervals, nuclet ~solated from the hvers, the two enzymes extracted and their activities assayed as described above The effect of aflatoxm B~ and stertgmatocystm on the acttvttles o f mouse hver nuclear DNAases Mice were dosed per os with toxin every second day for a period of 15 days. Aflatoxm was administered at a rate of 2 5 mg/kg body weight and sterlgmatocystm at a rate of 10 mg/kg Nuclei were prepared, the two enzymes extracted and their activities assayed as described above RESULTS
The m vltro effect of aflatoxm BI on the acttvtttes o f rat nuclear actd and alkahne DNAases No defimte activating or lnhlbatory effect of aflatoxxn B~ on the actlwtles of the two DNAases under m vztro conditions were observed The effects of aflatoxm B1 and sterigmatocystm on the acttvtttes o f actd and alkahne DNAases from rat hver nuclet Since we could not demonstrate any definite activating or inhibitory effect of aflatoxm B1 with m vttro experiments, tn vtvo studies were attempted as it is possible that aflatoxm B1 could be converted tn vtvo to the active toxin. In addition, the possible effect of stengmatocystln on these enzymes could only be investigated tn vtvo Chem-Btol Interacttons, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT D N A A S E S
357
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Fig 1 T h e m vtvo effects o f aflatoxm B 1 a n d s t e r l g m a t o c y s t m at dosing rates o f 1/5 L D s o on the acUvmes o f Wlstar rat hver nuclear D N A a s e s I a n d II over a period of 30 days D N A a s e I O, aftatoxm B1, A , sterlgmatocystln D N A a s e II O , aflatoxln B1, &, sterlgmatocystln Fig 2 T h e m vzvo effects o f aflatoxms BI a n d B 2 on the activities o f Wlstar rat liver nuclear D N A ases I a n d II over a period o f 18 days D N A a s e I 0 , aflatoxln B1, A , aflatoxln B2 D N A a s e II O , aflatoxm B I , A , aflatoxln B 2 F o r the dosing rates see MATERIALS AND METHODS
The results of the effects of aflatoxtn B1 and sterlgmatocystm on the actlvmes of the two DNAases over a period of 30 days are illustrated in Fig 1 and Table I From these data it seems that the activity of DNAase II, when rats were exposed to aflatoxln B1, Increased considerably No sigmficant effect of sterlgmatocysttn on the acttvmes of the two nuclear enzymes was observed
The influence of aflatoxm BE on the activities of the two nuclear DNAases Whereas aflatoxin Bt caused a substantial elevation in the activity of DNAase II, aflatoxln B2 had no effect on the activities of either enzyme (see Fig 2) This difference could be ascribed to the difference in the chemical structures of the toxins as the A2-bond of the terminal furane rmg of aflatoxln B2 is saturated (see Fig 3). Chem -Btol
Interactions,
3 (1971) 353-361
358
M . J . PITOUT~ H. A MCGEE, J C. SCHABORT
TABLE I THE EFFECTS OF AFLATOXIN B~ AND STERIGMATOCYSTINON ACID DNAAsE FROM RAT LIVER NUCLEAR SAP AT 1/5 L D s o DOSING OVER A 30-DAY PERIOD
Treatment
Day
Liver welght (g)
Total nuclear sap protetn (mg)
Total umts (U)
Spec acttwty Total mg ( U/mg prot ) protezn/g It ver
A~ C~ Sa
0
17 2 180 180
234 248 270
130 130 136
56 52 5 1
A C S
7
13 2 174 154
1 60 274 280
19 2 132 135
A C S
14
103 242 212
1 19 452 424
A C S
21
48 20 0 183
081 5 14 47
A
.
C S
30
.
21 7 180
. 44 46
U/g hver
0 1 4 ( 1 00) b 014(100) 0 1 5 (1 1)
076 072 076
12 0 48 48
0 12 (0 80) 0 1 6 (1 0) 018(11)
1 46 076 090
264 158 137
210 35 32
0 12 (06) 0 19(1 0) 0 2 0 ( 1 0)
264 065 065
184 14 4 109
227 2 75 211
0 17(07) 0 26 (1 0) 026(10)
383 0 72 060
021 025
057 075
. 124 134
.
.
28 29
A , C and S represent aflatoxin B~, control and sterlgmatocystln, respectively b The values m parentheses indicate the ratio obtained by dividing by C
The mfluence of aflatoxm B1 and stertgmatocystin on the acttvtty of the two DNAases from mouse hver In F~g 4 it can be seen that b o t h aflatoxm BI and sterlgmatocystln had n o effect on the activity of the a l k a h n e a n d acid D N A a s e s from mouse hver nuclm. Since the mouse in resistant to aflatoxm B1 carcinogenesis 14, the &fference m action of th~s
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Fig 3 Chemical formulae of aflatoxlns B~ and B 2 and sterlgmatocystln. Aflatoxins B1 and B e and sterlgmatocystln are represented by I, II and IH, respectively Fig 4 The m vlvo effects of aflatoxln B1 and sterlgmatocystln on the activities o f mouse liver nuclear D N A a s e s I and II over a period of 15 days Aflatoxln BI was dosed at a rate of 2 5 mg/kg body weight and sterlgmatocystln at a rate of 10 mg/kg D o s m g s were done every second day D N A a s e I O, aflatoxm B1, A , sterlgmatocystln D N A a s e II O, aftatoxxn B1, A, sterlgmatocystln
Chem.-Btol lnteracttons, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT
DNAAsES
359
toxin in the mouse could be attributed to a possible conversmn of aflatoxm Ba to a noncarcInogenlc substance(s) DISCUSSION
The relationship between aflatoxm B1 and the increased DNAase II activity could possibly be explained: (t) AftatoxIn B1, or a metabolic derivative, could interact in some way with the natural inhibitor of DNAase II to cause an increase in the activity of the enzyme It is known that mouse liver contains a protein which is capable of inhibiting DNAase II whether the protein is extracted from the same organ or obtained from other sources 24. (u) It has been shown that aflatoxln B~ inhibits the synthesis of only a few specific proteins, but total liver protein appears to be largely unaffected 25. The posslbihty, therefore, exists that there is a selective increase m DNAase II synthesis or a selective decrease m the synthesis of other nuclear sap proteins. Histologically distinct types of hepatic neoplasms are produced by aflatoxin B~ and sterigmatocystm; only aflatoxln B~ initially induces a marked prohferatlon of bile duct cells and subsequently bde duct epithelial adenomas and carcinomas 26,27 Variation in the level of DNAase II may be associated with the alterations in the amount of prohferatlng bile duct cells present in the tissue In contrast, sterlgmatocystln does not Induce a comparable degree of bile duct cell proliferation or ultimate bihary mahgnancy and no increase of DNAase II actwlty could be detected. An increased bile duct cell prohferatlon is not necessarily a specific effect of the toxin on this tissue, as the proliferation could be a futile attempt to re-estabhsh disrupted blle-canahcular potency in zones of hepatic parenchyma damaged by the toxin It is interesting to note that aflatoxln B1 has no effect on mouse liver bile duct cells 28. The exact function of acid DNAase in the nucleus is still unknown Recently, a considerable amount of data has been reported which indicates that DNAases are also involved in processes other than those which lead to extensive tissue breakdown29-aL These results demonstrate a close relationship between the activity of acid DNAase, the mitotic index and the speed of multiplication of liver, spleen, kidney and brain cells of rats killed at various times after birth It seems feasible that an abnormal activity of DNAase II could produce chromosome aberration and hence profound and Irreversible changes of the genetic expression which in turn could lead to the production of neoplastic cells. It is generally accepted ~4 that the mouse is resistant to aflatoxin B~ carcinogenesis. Therefore, the lack of effect of aflatoxln B~ on the activities of both nuclear DNAases from mouse hver could be attributed to the conversion of aflatoxln B~ to noncarcinogens 32, or to the lnabihty of aflatoxin B~ to react with the inhibitory protein of DNAase II. In either case, the lack of effect tends to confirm that nuclear acid DNAase plays some role, however minor, in the mechanism by which aflatoxin B1 produces cancer
Chem -Btol Interactions, 3 (1971) 353-361
360
M J PITOUT, H. A MCGEE, J. C. SCHABORT
ACKNOWLEDGEMENT
The authors are grateful to Dr I. F. H PURCHASE, South African Medical Research Council for helpful discussions and to Mr P. G KEMPFF for skilled assistance.
REFERENCES 1
M S MELZER, Effect o f carcinogens a n d other c o m p o u n d s on deoxyrlbonuclease, Btochlm
Blophys Acta, 138 (1967) 613-616 2
M S MELZER, Influence o f carcinogens a n d group-specific c o m p o u n d s on D N A a s e II, Can J
Bloehem, 47 (1968) 987-989 3
M J PITOUT, H A MCGEEANDJ C SCHABORT, T h e effect o f aflatoxlns Bl a n d B 2 a n d s t e r l g m a t o c y s t l n on nuclear D N A a s e s f r o m rat liver, Syrup on Mycotoxms m Human Health, Pretorla,
South Africa, 1970 4
5 6 7 8 9 10 ll 12 13 14 15 16
17
18 19 20 21 22
P LESCA, D TOUTAIN AND R TRUHAUT, A u g m e n t a t i o n tr6s pr6coce de l'actlvlte de la d6oxyrlbonucl6ase aclde p u l m o n a l r e de la sourls Swiss apr6s traltement p a r le benzo-3,4-pyr6ne, Compt Rend, Serte D, 268 (1969) 1238-1240 A C ALLISON AND G R PATON, C h r o m o s o m e d a m a g e In h u m a n diploid cells following activation o f lysosomal enzymes, Nature, 207 (1965) 1170-1172 A C ALLISON AND G R PATON, Lysosomes, c h r o m o s o m e s a n d cancer, Blochem J , 115 (1969) 31P-32P K F SWINGLE AND L J COLE, Acid deoxyrxbonuclease in rat liver cell nuclei isolated in the presence o f calcium ions, J Htstochem Cytochem, 12 (1964) 442-447 P LESCA, A g e variations o f acid deoxyrlbonuclease activity in m o u s e hver nuclei, Nature, 220 (1968) 76-77 K F SWINGLE,L J COLE AND J S BAILEY,Association o f neutral deoxyrlbonuclease with c h r o m a t l n isolated f r o m m a m m a l i a n cells,.Btochtm Btophys Acta, 149 (1967) 467-475 P J O'CONNOR, A n a l k a h n e deoxyrlbonuclease from rat hver n o n - h l s t o n e c h r o m a t m proteins, Btochem Btophys Res Commun, 35 (1969) 805-810 R HOWK AND T Y WANG, D N A polymerase from rat liver c h r o m o s o m a l proteins, Eur J Btochem, 13 (1970) 455-460 P J O'CONNOR AND S R AYAD, Some properties o f a n a l k a h n e d e o x y n b o n u c l e a s e f r o m regenerating rat liver, Btochtm Bzophys Acta, 217 (1970) 56-63 M S LEGATOR, Biological assay for aflatoxms, in L A GOLDBLATT (Ed.), Aflatoxln, Scientific Background, Control and Imphcattons, Academic Press, N e w York, 1969 W. H BUTLER,Aflatoxlns in laboratory animals, in L A GOLDBLATT ( E d ) , Aflatoxm, Scientific Background, Control and Imphcattons, A c a d e m i c Press, N e w York, 1969 M STEYN, R a p i d m e t h o d for the isolation o f pure aflatoxlns, J Ass Off Anal Chem, 53 (1970) 619-621 C W HOLZAPFEL,I F H PURCHASE,P S STEYN AND L GOUWS, T h e toxicity a n d chemical assay o f sterlgmatocystln, a carcinogenic m y c o t o x m , a n d ItS isolation f r o m two new fungal sources, S African M e d J , 40 (1966) 1100-1101 D A VAN DORP, A S M VAN DER ZIJDEN, R K BEERTHUIS, S SPARREBOOM,W O ORD, H DE IONGH AND R KEUNING, D l h y d r o a f l a t o x m B, a m e t a b o h t e o f A flavus, Rec Tray Chtm, 82 (1963) 587-592 R MAGGIO, P SIEKEWTZ AND G E PALADE,Studies on isolated nuclei, I Isolation a n d chemical characterization o f a nuclear fraction from guinea-pig liver, J Cell Btol, 18 (1963) 267-291 E BRESNICK AND A SCHWARTZ, Functional Dynamics o f the Cell, A c a d e m i c Press, N e w York, 1968 T Y. WANG, T h e Isolation properties a n d possible functions o f c h r o m a t m acidic proteins, J Biol Chem, 242 (1967) 1220-1226 M E HODES,L C YIP AND F R SANTOS, T h e purification a n d properties o f m o u s e liver deoxyrlbonuclease II, Enzymologta, 32 (1967) 241-255 O H LowRY, N J ROSEBROUGH,A L FARR AND R J RANDALL,Protein m e a s u r e m e n t with the F o h n p h e n o l reagent, J Btol Chem, 193 (1951) 265-275
Chem-Btol Interactions, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT D N A A S E S 23 24 25 26 27 28
29 30 31 32
361
I F H PURCHASE AND J J VAN DER WATT, A c u t e toxicity o f s t e r l g m a t o c y s t m to rats, Fd Cosmet Toxlcol, 7 (1969) 135-139 P LESCA AND C PAOLETTI, A protein Inhibitor o f acid d e o x y n b o n u c l e a s e s , Proc Natl Acad Sct ( U S ) , 64 (1969) 913-919 G N WOGAN, Biochemical responses to aflatoxms, Cancer Res, 28 (1968) 2282-2287 I F H PURCHASE AND J J VAN DER WATT, Carclnogenlclty o f sterlgmatocystm, Fd Cosmet Toxtcol, 8 (1970) 289-295 W H BUTLER AND J M BARNES, C a r c m o g e m c action o f g r o u n d n u t meal containing aflatoxm in rats, Fd Cosmet Toxwol, 6 (1968) 135-141 M N FERREIRA, Lesoes hepaticas cronlcas p r o v o c a d a s no ratinho pelas aflatoxmas B 1 e B 2 de a m e n d o l m c o n t a m l n a d o , Rewsta de Chntear Medzca, Umvers~dade de Lourenqo M a r q u e s , 1 0 9 6 8 ) 111-132 C CORDONNIER AND G BERNARDI, A c o m p a r a t w e study o f a o d D N A a s e s extracted f r o m different tissues a n d s p e o e s , Can J Btochem, 46 (1968) 989-995 I R LEHMAN, Deoxyrlbonucleases Their relationship to deoxyribonucleic acid synthesis, Ann Rev Btochem, 36 (1967) 645-668 P HOWARD-FLANDERS, D N A Repair, Ann Rev Blochem, 37 (1968) 175-200 M STEYN, M J PITOUT AND 1 F H PURCHASE, Brlt J Cancer, In the press
Chem-Btol Interacttons, 3 (1971) 353-361
353
Elsevier Pubhshmg Company, Amsterdam Printed m The Netherlands
T H E E F F E C T OF A F L A T O X I N B1, A F L A T O X I N B2 A N D S T E R I G M A T O CYSTIN ON NUCLEAR DEOXYRIBONUCLEASES FROM RAT AND M O U S E LIVERS
M J PITOUT a, H A M c G E E a AND J C SCHABORT u
aDlvtston of Toxicology, National Institute for Nutritional Diseases, S A Medical Research Council, Pretorta, and t, Department of Biochemistry, Rand AJmkaans University, Johannesburg (Repubhc of South Africa) (Recewed December 4th, 1970) (Accepted January 29th, 1971)
SUMMARY
Certain carcinogens have an effect on the activity of pancreatic deoxynbonuclease I (DNAase I, EC 3 1 4 5) The effect of two potent mycocarclnogens, vtz. aflatoxln B1 and stengmatocystm, as well as the weak carcinogen, aflatoxm B2, on the activity of two nuclear DNAases (DNAases I and II) from rat hver was therefore investigated. No significant interaction between aflatoxm B1 and the two enzymes was observed. Stengmatocystm ts almost insoluble in aqueous solutions and therefore could not be used for m v i t r o studies. I n v t v o experiments, however, clearly indicated a marked effect of aflatoxln BI on the total activity of DNAase II. F r o m these results it is suggested that aflatoxm B1 could be a precarcmogen and that it may be converted, probably by mlcrosomal enzymes, to the active carcinogenic compound. Alternatively, aflatoxm B1 could react with the natural inhibitor, resulting in higher enzyme activity In the normal cell, activity of the enzyme must be under rigorous control in order top reserve the integrity of the genetic message Abnormal circumstances, causing release of activity, might result in anomalies in the genetic expression mechanism. The negative result obtained with aflatoxln B2 illustrates the fact that the double-bond of the terminal furane ring plays a vital role in determining the toxic and carcinogenic activity of aflatoxm B 1
Stengmatocystin produced no effect on either enzyme, suggesting that the mechanisms of carcinogenic action of the two toxins are different. The fact that orally dosed aflatoxln B1 has no effect on the activity of DNAase II from mouse liver nuclei could support the concept that the mouse is capable of convertmg the aflatoxln B~ to noncarcmogenlc metabohtes Abbreviation DMSO, dlmethylsulphoxlde.
Chem-Btol Interactions, 3 (1971) 353-361
354
M J. PITOUT, H A MCGEE, J. C. SCHABORT
INTRODUCTION
There have been a number of m w t r o and m v t v o studies on the effects of various potent carcinogens such as the butter yellow-type carcinogens, urethane, y-butyrolactone and dtepoxybutane on the activity of pancreatxc DNAase I. In these studies, considerable mcreases in activity were found as a result of treatment with the carcmogens 1-3. With weak carcinogens no activation of pancreatic DNAase I was observed The potent carcinogens have been found to be devoid of any effect on the actlvlty of spleen DNAase II. However, LESCAe t a l 4 demonstrated decisively a sharp rise in pulmonary DNAase II activity when rats were treated with the carcinogen benz-3,4-pyrene. It is widely, although not umversally, beheved that the basic defect in cancer is a chromosomal mutation in somatic cells. Evidence suggesting a relationship between lysosomal damage and the occurrence of chromosomal aberrations has been presented and it was, therefore, suggested that the released lysosomal DNAase might be able to penetrate the nucleus and attack the chromosomes 5,6 Although the presence of an acid DNAase (DNAase type II) in nuclei from various sources has been shown by several workers 7,8, the enzyme has never been isolated and neither has any function been ascribed to it Apart from DNAase II, alkahne DNAases (type I) which are associated with the non-hlstone fraction of chromatm have only recently been described 9-12 In the hght of the above-mentioned observations, It was decided to investigate the possible influence of the hepatocarclnogens aflatoxln B1 and sterlgmatocystm on the actlvRles of the two DNAases from rat liver. In addition, the effect of aflatoxm Be on the actlwtles of the two nuclear DNAases from rat liver was also studied Ariatoxin B2 has a slmdar molecular structure to aflatoxm B1, but it has a relatively weak toxic and carcinogenic action m rats in comparison to aflatoxln B1, the most potent carcinogen known ~3 For the sake of comparison, the effect of aflatoxm Bt and sterlgmatocystln on the activity of acid and alkaline DNAases from mouse liver nuclei was also investigated It is known that the mouse is resistant to aflatoxm B1 carcinogenesis t4. MATERIALS AND METHODS
Ammals
Random-bred male Wlstar-denved rats with an average body weight of 200 4- 10 g and male albino mice (22 :~ 2 g body weight) were used. The mice were obtained from Onderstepoort Veterinary Research Institute, Pretorm, which originally obtained them from the Wellcome Bureau of Scientific Research, London, England. Rats and mice were fed a balanced pellet diet, and were provided with tap water a d h b t t u m Rats and mice were divided into 3 groups, t.e aflatoxln B1 and stengmatocystm treated rats and the control group, respectively Each group consisted of 3 rats or 4 mice. DMSO was used as solvent for the toxins and the control animals were dosed only with DMSO Chem-Btol Interactions, 3 (1971) 353-361
EFFECT OF CARCINOGENSON MOUSEAND RAT DNAASES
355
Chemicals Thymus-gland D N A was obtamed from Sigma Chemical C o , Ohio, U.S.A All other chemicals used were of analytical reagent grade Aflatoxm B1 and stengmatocystm were isolated and purified according to STEYNis and HOLZAPFEL et al a6, respectively Aflatoxm B2 was prepared from aflatoxm B1 by means of hydrogenation according to VAN DORP et al 1 7 Extractton and tsolatton of nuclei from mouse and rat hver All operations were done in the cold (2-5 °) The nuclei were prepared according to the method of MAGGIO et al 18 with minor alterations The hvers of each group were pooled and homogenized m 0 88 M sucrose-3 m M MgC12 solution in the ratio of 10-15 g hver/100 ml sucrose solution The suspension was filtered through 4 layers of cheesecloth muslin gauze and centrifuged at 1500 × g for 10 mm The precipitate was suspended in 2 3 M sucrose-3 m M MgCI2 solution and the supernatant was recentrlfuged as described above The process was repeated twice To separate the nuclei from contaminants, the 2 3 M sucrose suspension (180 ml/10-15 g liver) was centrifuged at 44 000 < g for 65 m m The purity of the nuclei was checked by electron and light microscopy 19. Extraction o f the acid and alkahne DNAases from rat and mouse hver nuclet Since previous studies on the effect of carcinogens on DNAases were done m vttro (see refs 1-3), an attempt was made to isolate and purify these enzymes from rat liver nuclei 3 Of the two enzymes, the acid DNAase was found to be the more stable enzyme, and was, therefore, purified to a greater extent 3 For the in VlVO experiments, however, crude nuclear DNAases from rat and mouse livers were prepared by the following procedure: To lyse the nuclei, they were suspended in 5 ml of 0 05 M citrate-0 005 M mercaptoethanol buffer (pH 7.0) Centrlfugatlon at 2000 × g for 20 rain yielded a preclpitate (chromatm) and the supernatant (nuclear sap) The chromatln acidic proteins were obtained by the procedure of WANG2° The p H of the nuclear sap was carefully adjusted to 4.7 by the addition of acetic acid, and the resultant milky suspension was clarified by centnfugatlon at 10 000 × g for 20 m m to give a clear, colourless supernatant which contained the acid DNAase. Assay for acttvmes o f nuclear DNAases The activities of acid and alkahne DNAases were assayed according to HODES et al 21 and O'CONNOR 1°, respectively For both enzymes, one unit (U) of enzyme is that amount of enzyme which produces one AA26o umt/h. Specific actlvxty is expressed as unlts/mg protein Protein was determined according to the method of LOWRY et al. 2z The m vitro effect o f aflatoxm B1 on the acttvttles of actdandalkahne DNAases from rat hver In vttro investigations could not be performed with ster~gmatocystm due to its very poor solublhty A stock solution of aflatoxm B1 was made in 1,2-propylene Chem-Btol Interactions, 3 (1971) 353-361
356
M . J . PITOUT~ H A. MCGEE~ J C. SCHABORT
glycol (7 mg/ml) and different concentrations of aflatoxm B~ were prepared from the stock solution by dilution (0.2 ml aflatoxm B1 solution added to the incubation mxxture) The activities of the two nuclear DNAases in the presence of different concentratlons of aflatoxm B1 were measured as described above. Control values were obtained by using 0 2 ml of propylene glycol In vtvo effects of aflatoxm B~ and stertgmatocystm on the acttvtttes of actd and alkahne DNAases from rat hver nuclet Wlstar rats were given a dally per os dose over a 30-day period. The dady dose was eqmvalent to 1/5 LDso of aflatoxm B 1 and stengmatocystln, where the LDso values of aflatoxln B~ and sterlgmatocystln were taken as 7 mg/kg and 160 mg/kg body weight, respectively 13'23 Rats were kdled by decapitation at predetermined time intervals, the hvers excised, nuclei xsolated, the two enzymes extracted and their activities assayed as described above The effect o f aflatoxm B 2 o n the acttvtttes of the two nuclear DNAases The similarity in chemical structure between aflatoxins B1 and B2 prompted an m vtvo study of the effect of aflatoxm B2 on the actlwtles of the acid and alkaline DNAases Male Wlstar rats were dosed with aflatoxlns B~ and B2 at 1 75 mg/kg dady over a period of 18 days. Rats were kdled at predetermined time intervals, nuclet ~solated from the hvers, the two enzymes extracted and their activities assayed as described above The effect of aflatoxm B~ and stertgmatocystm on the acttvttles o f mouse hver nuclear DNAases Mice were dosed per os with toxin every second day for a period of 15 days. Aflatoxm was administered at a rate of 2 5 mg/kg body weight and sterlgmatocystm at a rate of 10 mg/kg Nuclei were prepared, the two enzymes extracted and their activities assayed as described above RESULTS
The m vltro effect of aflatoxm BI on the acttvtttes o f rat nuclear actd and alkahne DNAases No defimte activating or lnhlbatory effect of aflatoxxn B~ on the actlwtles of the two DNAases under m vztro conditions were observed The effects of aflatoxm B1 and sterigmatocystm on the acttvtttes o f actd and alkahne DNAases from rat hver nuclet Since we could not demonstrate any definite activating or inhibitory effect of aflatoxm B1 with m vttro experiments, tn vtvo studies were attempted as it is possible that aflatoxm B1 could be converted tn vtvo to the active toxin. In addition, the possible effect of stengmatocystln on these enzymes could only be investigated tn vtvo Chem-Btol Interacttons, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT D N A A S E S
357
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Fig 1 T h e m vtvo effects o f aflatoxm B 1 a n d s t e r l g m a t o c y s t m at dosing rates o f 1/5 L D s o on the acUvmes o f Wlstar rat hver nuclear D N A a s e s I a n d II over a period of 30 days D N A a s e I O, aftatoxm B1, A , sterlgmatocystln D N A a s e II O , aflatoxln B1, &, sterlgmatocystln Fig 2 T h e m vzvo effects o f aflatoxms BI a n d B 2 on the activities o f Wlstar rat liver nuclear D N A ases I a n d II over a period o f 18 days D N A a s e I 0 , aflatoxln B1, A , aflatoxln B2 D N A a s e II O , aflatoxm B I , A , aflatoxln B 2 F o r the dosing rates see MATERIALS AND METHODS
The results of the effects of aflatoxtn B1 and sterlgmatocystm on the actlvmes of the two DNAases over a period of 30 days are illustrated in Fig 1 and Table I From these data it seems that the activity of DNAase II, when rats were exposed to aflatoxln B1, Increased considerably No sigmficant effect of sterlgmatocysttn on the acttvmes of the two nuclear enzymes was observed
The influence of aflatoxm BE on the activities of the two nuclear DNAases Whereas aflatoxin Bt caused a substantial elevation in the activity of DNAase II, aflatoxln B2 had no effect on the activities of either enzyme (see Fig 2) This difference could be ascribed to the difference in the chemical structures of the toxins as the A2-bond of the terminal furane rmg of aflatoxln B2 is saturated (see Fig 3). Chem -Btol
Interactions,
3 (1971) 353-361
358
M . J . PITOUT~ H. A MCGEE, J C. SCHABORT
TABLE I THE EFFECTS OF AFLATOXIN B~ AND STERIGMATOCYSTINON ACID DNAAsE FROM RAT LIVER NUCLEAR SAP AT 1/5 L D s o DOSING OVER A 30-DAY PERIOD
Treatment
Day
Liver welght (g)
Total nuclear sap protetn (mg)
Total umts (U)
Spec acttwty Total mg ( U/mg prot ) protezn/g It ver
A~ C~ Sa
0
17 2 180 180
234 248 270
130 130 136
56 52 5 1
A C S
7
13 2 174 154
1 60 274 280
19 2 132 135
A C S
14
103 242 212
1 19 452 424
A C S
21
48 20 0 183
081 5 14 47
A
.
C S
30
.
21 7 180
. 44 46
U/g hver
0 1 4 ( 1 00) b 014(100) 0 1 5 (1 1)
076 072 076
12 0 48 48
0 12 (0 80) 0 1 6 (1 0) 018(11)
1 46 076 090
264 158 137
210 35 32
0 12 (06) 0 19(1 0) 0 2 0 ( 1 0)
264 065 065
184 14 4 109
227 2 75 211
0 17(07) 0 26 (1 0) 026(10)
383 0 72 060
021 025
057 075
. 124 134
.
.
28 29
A , C and S represent aflatoxin B~, control and sterlgmatocystln, respectively b The values m parentheses indicate the ratio obtained by dividing by C
The mfluence of aflatoxm B1 and stertgmatocystin on the acttvtty of the two DNAases from mouse hver In F~g 4 it can be seen that b o t h aflatoxm BI and sterlgmatocystln had n o effect on the activity of the a l k a h n e a n d acid D N A a s e s from mouse hver nuclm. Since the mouse in resistant to aflatoxm B1 carcinogenesis 14, the &fference m action of th~s
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Fig 3 Chemical formulae of aflatoxlns B~ and B 2 and sterlgmatocystln. Aflatoxins B1 and B e and sterlgmatocystln are represented by I, II and IH, respectively Fig 4 The m vlvo effects of aflatoxln B1 and sterlgmatocystln on the activities o f mouse liver nuclear D N A a s e s I and II over a period of 15 days Aflatoxln BI was dosed at a rate of 2 5 mg/kg body weight and sterlgmatocystln at a rate of 10 mg/kg D o s m g s were done every second day D N A a s e I O, aflatoxm B1, A , sterlgmatocystln D N A a s e II O, aftatoxxn B1, A, sterlgmatocystln
Chem.-Btol lnteracttons, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT
DNAAsES
359
toxin in the mouse could be attributed to a possible conversmn of aflatoxm Ba to a noncarcInogenlc substance(s) DISCUSSION
The relationship between aflatoxm B1 and the increased DNAase II activity could possibly be explained: (t) AftatoxIn B1, or a metabolic derivative, could interact in some way with the natural inhibitor of DNAase II to cause an increase in the activity of the enzyme It is known that mouse liver contains a protein which is capable of inhibiting DNAase II whether the protein is extracted from the same organ or obtained from other sources 24. (u) It has been shown that aflatoxln B~ inhibits the synthesis of only a few specific proteins, but total liver protein appears to be largely unaffected 25. The posslbihty, therefore, exists that there is a selective increase m DNAase II synthesis or a selective decrease m the synthesis of other nuclear sap proteins. Histologically distinct types of hepatic neoplasms are produced by aflatoxin B~ and sterigmatocystm; only aflatoxln B~ initially induces a marked prohferatlon of bile duct cells and subsequently bde duct epithelial adenomas and carcinomas 26,27 Variation in the level of DNAase II may be associated with the alterations in the amount of prohferatlng bile duct cells present in the tissue In contrast, sterlgmatocystln does not Induce a comparable degree of bile duct cell proliferation or ultimate bihary mahgnancy and no increase of DNAase II actwlty could be detected. An increased bile duct cell prohferatlon is not necessarily a specific effect of the toxin on this tissue, as the proliferation could be a futile attempt to re-estabhsh disrupted blle-canahcular potency in zones of hepatic parenchyma damaged by the toxin It is interesting to note that aflatoxln B1 has no effect on mouse liver bile duct cells 28. The exact function of acid DNAase in the nucleus is still unknown Recently, a considerable amount of data has been reported which indicates that DNAases are also involved in processes other than those which lead to extensive tissue breakdown29-aL These results demonstrate a close relationship between the activity of acid DNAase, the mitotic index and the speed of multiplication of liver, spleen, kidney and brain cells of rats killed at various times after birth It seems feasible that an abnormal activity of DNAase II could produce chromosome aberration and hence profound and Irreversible changes of the genetic expression which in turn could lead to the production of neoplastic cells. It is generally accepted ~4 that the mouse is resistant to aflatoxin B~ carcinogenesis. Therefore, the lack of effect of aflatoxln B~ on the activities of both nuclear DNAases from mouse hver could be attributed to the conversion of aflatoxln B~ to noncarcinogens 32, or to the lnabihty of aflatoxin B~ to react with the inhibitory protein of DNAase II. In either case, the lack of effect tends to confirm that nuclear acid DNAase plays some role, however minor, in the mechanism by which aflatoxin B1 produces cancer
Chem -Btol Interactions, 3 (1971) 353-361
360
M J PITOUT, H. A MCGEE, J. C. SCHABORT
ACKNOWLEDGEMENT
The authors are grateful to Dr I. F. H PURCHASE, South African Medical Research Council for helpful discussions and to Mr P. G KEMPFF for skilled assistance.
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Chem-Btol Interactions, 3 (1971) 353-361
EFFECT OF CARCINOGENS ON MOUSE AND RAT D N A A S E S 23 24 25 26 27 28
29 30 31 32
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Chem-Btol Interacttons, 3 (1971) 353-361