Chem.J?iol. Interactions, 10 (1975) 343-347 0 Elsevier sfieatific Publishing Company,Amsterdam-
C~ARA~ERIZATION
OF ~DNOSOM~
Printedin The Netherlands
343
PRODUCED BY AFLATOXIN Bi
LINDA C. HAYES, FRED V. PLAPP, LOWELL L. TILZER AND MASAHIRO CHIGA Department of Pathology and Oncology, Urtlversity of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kan. 66103 (U.S.A.) (Received July IS& 1974) (Revision m&ed October 29th, 1974)
SUMMARY
A single injection of 1.5 mg aflatoxin Bl per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S and 60 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the j~onosomes was calculated to be 0.6, indicting 0.6 tRNA and/or aminoacy~ tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of aflatoxin Bl produces monosomes which resemble runoff ribosomes.
INTRODUCTION
Aflatoxin 31, an hepatotoxic and hepatocarcinogenic metabolite produced by Aspergi~~~s~u~~us, has been shown to inhibit protein synthesislvs and to cause polysome di~~~gation~~4 in v&o in the rat. However, the m~hanism of action of aflatoxin BI
in inhibiting protein synth~is is unknown. It has been postulated that the inhibition of protein synthesis by a~atoxin & beyond 7 h post injection might be due to impairment of transcription%. In this case the monosomes produced by aflatoxin BI should resemble runoff ribosomes. In order to further elucidate the mechanism by which aflatoxin BI disaggregates liver polysomes, we studied the dissociability and tRNA content of the monosomes produced by this mycotoxin since we have previously shown that these parameters can determine whether monosomes are complexed, runoff, or falloff ribosomess-7. This in~~ti~tion
was supported in pan by NlH grant SlBl GM 1783 and a
Kaw Valley Heart Association.
grant from the
344 MATERIALS AND METHODS
Disaggregation of polysomes
Inbred male F-344 rats weighing 150 to 200 g were given a single intraperitoneal injection of 1.5 mg aflatoxin Bl (Makor Biochemicals Ltd. of Jerusalem) per kg body weight, suspended in 0.2 ml of propylene glycol. The animals were allowed free access to water and Purina laboratory chow and consumed approx. 8 g of chow per 100 g body weight before sacrifice 18 h later. Control rats were given a single intraperitoneal injection of 0.2 ml of propylene glycol and starved for 18 h. The livers from experimental and control rats were removed, weighed, and homogenized with 3 vol. of TKMS buffer containing 10 mM Tris-HCI (pH 7.1), 25 mM KCI, 5 mM MgS04, and 0.25 M sucrose in a Potter-Elvehjem homogenizer. This homogenate was centrifuged at IO 000 x g for 10 min. A 10% aqueous solution of sodium deoxycholate was added to the postmitochondrial supernatant to a final concentration of 1%. 0.2 ml of this solution was layered on 0.5 to 1.2 M linear sucrose gradients and centrifuged for 90 min at 114 000 x g in a Beckman L5-65 centrifuge with a type SW 50.1 rotor. The gradients were scanned at 260 nm in a Gilford spectrophotometer equipped with a flow cell. Dissociation of monosomes
Ribosome pellets were prepared from the postmitochondrial supernatant as previously described* and resuspended in TKM buffer containing 0.3 M KCI. 0.2 ml of this solution was layered on 0.5 to 1.2 M linear sucrose gradients containing 0.3 M KCI TKM buffer. The gradients were centrifuged for 4 h at 114 000 x g and then scanned at 260 nm as described above. Transfer RNA con tent
To determine the tRNA content of the monosomes produced by afiatoxin BI, the 80 S monosome peak was isolated from sucrose gradients and tibosome particles were precipitated by the method of Dessev and Grancharovs. The precipitate was resuspended in 5 mM Tris-MCI (pH 7.4) and divided into two equal fractions. One fraction was incubated with 3 mg/ml Pronase (Calbiochem, B grade) for 60 min at 37” in the presence of 4 mg/ml sodium dodecyl sulfate while the other fraction was untreated. Both fractions were then extracted with chloroform-isoamyl alcohol0 and RNA was precipitated by making the aqueous phase 0.2 M with respect to NaCI, mixing with two volumes of 95 % ethanol, and placing the solution at -20” overnight. 350 pg of RNA was layered on 4j‘/, polyacrylamide gels and electrophoresed for 70 min at 5 mA per gel in 36 mM Tris-phosphate (pH 7.8), 30 mM NasHP04, and 1 mM EDTA buffer as described by Kabat 10. Gels were scanned at 260 nm in a Gilford spectrophotometer equipped with a linear transporter. E. co/i tRNA was used to estimate the position of the 4 S and 5 S RNA peaks. The percentage of acrylamide in the gel and the conditions of electrophoresis were such that 28 S and 18 S RNA remained at the top of the gel while 5 S and 4 S RNA readily penetrated. Since each ribosome contains one 5 S RNA molecule, the
345 4 S to 5 S molar RNA ratio is an estimate of the amount of 4 S RNA present per
ribosome. To calculate this ratio the 4 S and 5 S RNA peaks were traced from the spectrophotometric scan, cut out, and weighed. The 4 S value was multiplied by 1.5 and divided by the 5 S value to obtain the 4 S to 5 S molar RNA ratio as described by Kabatl*. RESULTS
Disaggregationof poiysomes Fig. IA shows that injection of propylene glycol alone followed by 18 h of sedation had no effect on the rat liver polysomes. Fig. I B demonst~t~ that a single dose of 1.5 mg aflatoxin A per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomcs 18 h after injection. Dissociutionof mom.womes Inspection of Fig. lC demonstrate that the 80 S monosom~ dissociated into 40 S and 60 S ribosomal subunits when centrifuged in linear sucrose gradients containing 0.3 M KCI. Hence, the monosomes produced by aflatoxin & dissociate into subunits similar to monosomes which are free of messenger RNA+‘.
It has been shown previously s-r.10 that incubation with Pronase prior to RNA extraction permits all types of tRNA (tRNA, aminoacyl tRNA, and peptidyl tRNA) to be extracted since nascent peptides are removed from peptidyl tRNA. Extraction of RNA without prior Pronase incubation extracts only RNA originally present as tRNA and aminoacyl tRNA.
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2 3
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8
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Fig. 1. Ri~om~p~lysom~ pro5les of rat livers in 0.5 to I .2 M linear sucrosegradients. The arrow indicates the position of the 808 peak. A, Controi ri~o~~l~o~ profile, ~tmit~hondrial supernatant, centrifuged 90 min: B and C, 18 h aRer a single intraperitoneal injection of 1.5 mg aflatoxin BI per kg body weight; B, postmitochondrial supernatant, centrifuged 90 min; C, ribosome pellet resuspendedin 10 mM Tris-HCI (pH 7.1), 0.3 M KC4 and 5 mM MgSOa centrifuged 240 min.
TABLE I TRANSFER RNA CONTRNT OF RIROSOMRS
4s:ss mtio with Ptvnase 1.7 (1) Normal polysomal ribosomes (2) Control ribosomes(propyleneglycol 1.7 + 18 h starvation) 0.7 (3) Starvation monosomes 0.6 (4) Aflatoxin B1 monosomes
4s:ss ratio without Prottase 0.9 0.9 0.7 0.6
Table I, line 1, demonstrates the tRNA content of normal polysomes. When ribosomes were incubated with Pronase the 4 S to 5 S RNA ratio was 1.7; but when ribosomes were extracted without Pronase treatment the 4 S to 5 S RNA ratio decreased to 0.9. Thus, normal ribosomes contain 0.9 molecule of tRNA and/or aminoacyl tRNA and 0.8 molecule of peptidyl tRNA. As seen in Table I, line 2, the ribosomes from control rats have a 4 S to 5 S RNA ratio of 1.7 when incubated with Psonase and a ratio of 0.9 without Pronase treatment. Thus control ribosomes do not differ from normal ribosomes in their tRNA content. Table I, line 3, reveals that monosomes produced by starvation of mice for 72 h contain 0.7 molecule tRNA when incubated with Pronase and without Pronase treatment. This implies that these starvation monosomes contain 0.7 molecule of tRNA and/or aminoacyl tRNA but lack peptidyl tRNAsB7. Similarly, as seen in Table I, line 4, the monosomes produced by aflatoxin A contain 0.6 molecule of tRNA with and without Pronase treatment. This result indicates that the aflatoxin monosomes resemble monosomes produced by starvation and contain 0.6 molecule of tRNA and/or aminoacyl tRNA but no peptidyl tRNA. DISCUssxON
We have shown in agreement with others s-4 that a single injection of aflatoxin Br disaggregates rat liver polysomes into monosomes within 18 h. It has been previously shown that aflatoxin Br inhibits nucleoplasmic RNA polymerase Btr which is presumably responsible for messenger RNA synthesisl*. Inhibition of messenger RNA synthesis could account for the decreased amount of liver polysomes seen witllin 18 h after injection of aflatoxin Bl. These results are consistent with our finding that the monosomes produced by atlatoxin Br dissociated into subunits in buffer containing 0.3 M KCI, indicating that they lack messenger RNAE-‘. We also found that these monosomes contained less than one molecule of tRNA and/or aminoacyl tRNA, but no prptidyl tRNA, similar to the monosomes produced by starvation. The observations that the monosomes produced by aflatoxin Bl are dissociable into subunits and lack peptidyl tRNA suggest that they are runoff ribosomessJ.
347 REFERENCES 1 S. Villa-Tnvino and D. D. Leaver, Effects of the hepatotoxic agents retrorsine and a&toxin BJ on hepatic protein synthesii in the rat, B&hem. J., 109 (1968) 87-91. 2 A. Sarasin and Y. Mottle, In viva elfect of a&toxin L on protein synthesii in rat liver, FEB&’ Lerrers, 29 (1973) 329-332. 3 A. K. Roy, ES&s of aflatoxin BI on polysomal profiles and RNA synthesis in rat Iiver, B&h/m. Biophys. Actu, 169 (1969) 206211. 4 R. S. Pang and 0. N. Wopan, Tie course of alterations of rat liver pofysome protiles induced by aflatoxin &, B&hem. Phtwwcol., 18 (1969) 2357-2361. 5 F, V. Plapp and M. Chiga, Dissociation of ribosomes produced by dimethylnitrosamine and Iasiocarpine in mouse liver, l&&em. Phurmao~., 22 (1973) 1681-1682. 6 F. V. Plapp, L. C. Hayes, L. Tilzer and hi. Chiga, ~~iabiIity and transfer RNA content of rno~ produced by di~hyIni~mi~ and starvation, Narurp, 247 (1974) 311-313. 7 L. L. Tilzer, F. V. Plapp, L. C!. Hayes, and M. ChQa, Certain &aracteristics of monosomes produced by CC%, Biochem, PlwmuMof., (in press). 8 G. N. Dessev and K. Grancharov, Precipitation of RNA, DNA, and nucleoprotein particles from very dilute solutions, Anal. Biochem., 53 (1973) 269-271. 9 K. Oda and W. K. Joklik, Hybridization and sedimentution studies on “early” and “late” vaccinia messenger RNA, J. Mol. Biol., 27 (1964) 395-419. 10 D. Kabat, Direct method of measuring transfer ribonucleic acid binding to polyribosomes and to single ribosomes in ceils of higher organisms, Anal. B&hem., 39 (1971) 228-242. 11 E. 0. Akinrimisi, B. J. Benecke and K. H. Seifart, Inhibition of rat-liver RNA polymerase in vitro by aftatoxin Bl in the presence of a microsomal fraction, Europwn J. Bio&vn., 42 (19741 333-339. 12 M. B. Mathews, Mammalian messenger RNA, in P. N. Campbell and F. Dickens @is.), Es%zyr in B~he/~~is$ry, Academb Press, New York, 9, 1973, pp. 59402.
C~ARA~ERIZATION
OF ~DNOSOM~
Printedin The Netherlands
343
PRODUCED BY AFLATOXIN Bi
LINDA C. HAYES, FRED V. PLAPP, LOWELL L. TILZER AND MASAHIRO CHIGA Department of Pathology and Oncology, Urtlversity of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kan. 66103 (U.S.A.) (Received July IS& 1974) (Revision m&ed October 29th, 1974)
SUMMARY
A single injection of 1.5 mg aflatoxin Bl per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S and 60 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the j~onosomes was calculated to be 0.6, indicting 0.6 tRNA and/or aminoacy~ tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of aflatoxin Bl produces monosomes which resemble runoff ribosomes.
INTRODUCTION
Aflatoxin 31, an hepatotoxic and hepatocarcinogenic metabolite produced by Aspergi~~~s~u~~us, has been shown to inhibit protein synthesislvs and to cause polysome di~~~gation~~4 in v&o in the rat. However, the m~hanism of action of aflatoxin BI
in inhibiting protein synth~is is unknown. It has been postulated that the inhibition of protein synthesis by a~atoxin & beyond 7 h post injection might be due to impairment of transcription%. In this case the monosomes produced by aflatoxin BI should resemble runoff ribosomes. In order to further elucidate the mechanism by which aflatoxin BI disaggregates liver polysomes, we studied the dissociability and tRNA content of the monosomes produced by this mycotoxin since we have previously shown that these parameters can determine whether monosomes are complexed, runoff, or falloff ribosomess-7. This in~~ti~tion
was supported in pan by NlH grant SlBl GM 1783 and a
Kaw Valley Heart Association.
grant from the
344 MATERIALS AND METHODS
Disaggregation of polysomes
Inbred male F-344 rats weighing 150 to 200 g were given a single intraperitoneal injection of 1.5 mg aflatoxin Bl (Makor Biochemicals Ltd. of Jerusalem) per kg body weight, suspended in 0.2 ml of propylene glycol. The animals were allowed free access to water and Purina laboratory chow and consumed approx. 8 g of chow per 100 g body weight before sacrifice 18 h later. Control rats were given a single intraperitoneal injection of 0.2 ml of propylene glycol and starved for 18 h. The livers from experimental and control rats were removed, weighed, and homogenized with 3 vol. of TKMS buffer containing 10 mM Tris-HCI (pH 7.1), 25 mM KCI, 5 mM MgS04, and 0.25 M sucrose in a Potter-Elvehjem homogenizer. This homogenate was centrifuged at IO 000 x g for 10 min. A 10% aqueous solution of sodium deoxycholate was added to the postmitochondrial supernatant to a final concentration of 1%. 0.2 ml of this solution was layered on 0.5 to 1.2 M linear sucrose gradients and centrifuged for 90 min at 114 000 x g in a Beckman L5-65 centrifuge with a type SW 50.1 rotor. The gradients were scanned at 260 nm in a Gilford spectrophotometer equipped with a flow cell. Dissociation of monosomes
Ribosome pellets were prepared from the postmitochondrial supernatant as previously described* and resuspended in TKM buffer containing 0.3 M KCI. 0.2 ml of this solution was layered on 0.5 to 1.2 M linear sucrose gradients containing 0.3 M KCI TKM buffer. The gradients were centrifuged for 4 h at 114 000 x g and then scanned at 260 nm as described above. Transfer RNA con tent
To determine the tRNA content of the monosomes produced by afiatoxin BI, the 80 S monosome peak was isolated from sucrose gradients and tibosome particles were precipitated by the method of Dessev and Grancharovs. The precipitate was resuspended in 5 mM Tris-MCI (pH 7.4) and divided into two equal fractions. One fraction was incubated with 3 mg/ml Pronase (Calbiochem, B grade) for 60 min at 37” in the presence of 4 mg/ml sodium dodecyl sulfate while the other fraction was untreated. Both fractions were then extracted with chloroform-isoamyl alcohol0 and RNA was precipitated by making the aqueous phase 0.2 M with respect to NaCI, mixing with two volumes of 95 % ethanol, and placing the solution at -20” overnight. 350 pg of RNA was layered on 4j‘/, polyacrylamide gels and electrophoresed for 70 min at 5 mA per gel in 36 mM Tris-phosphate (pH 7.8), 30 mM NasHP04, and 1 mM EDTA buffer as described by Kabat 10. Gels were scanned at 260 nm in a Gilford spectrophotometer equipped with a linear transporter. E. co/i tRNA was used to estimate the position of the 4 S and 5 S RNA peaks. The percentage of acrylamide in the gel and the conditions of electrophoresis were such that 28 S and 18 S RNA remained at the top of the gel while 5 S and 4 S RNA readily penetrated. Since each ribosome contains one 5 S RNA molecule, the
345 4 S to 5 S molar RNA ratio is an estimate of the amount of 4 S RNA present per
ribosome. To calculate this ratio the 4 S and 5 S RNA peaks were traced from the spectrophotometric scan, cut out, and weighed. The 4 S value was multiplied by 1.5 and divided by the 5 S value to obtain the 4 S to 5 S molar RNA ratio as described by Kabatl*. RESULTS
Disaggregationof poiysomes Fig. IA shows that injection of propylene glycol alone followed by 18 h of sedation had no effect on the rat liver polysomes. Fig. I B demonst~t~ that a single dose of 1.5 mg aflatoxin A per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomcs 18 h after injection. Dissociutionof mom.womes Inspection of Fig. lC demonstrate that the 80 S monosom~ dissociated into 40 S and 60 S ribosomal subunits when centrifuged in linear sucrose gradients containing 0.3 M KCI. Hence, the monosomes produced by aflatoxin & dissociate into subunits similar to monosomes which are free of messenger RNA+‘.
It has been shown previously s-r.10 that incubation with Pronase prior to RNA extraction permits all types of tRNA (tRNA, aminoacyl tRNA, and peptidyl tRNA) to be extracted since nascent peptides are removed from peptidyl tRNA. Extraction of RNA without prior Pronase incubation extracts only RNA originally present as tRNA and aminoacyl tRNA.
C
oTOP
I
2
3 4 3
TOP
’
s
I
2 3
’
EFFLUENT,
8
J
4
3
TOP
f
t
I
2 3
m
I
,
4 5
mI
Fig. 1. Ri~om~p~lysom~ pro5les of rat livers in 0.5 to I .2 M linear sucrosegradients. The arrow indicates the position of the 808 peak. A, Controi ri~o~~l~o~ profile, ~tmit~hondrial supernatant, centrifuged 90 min: B and C, 18 h aRer a single intraperitoneal injection of 1.5 mg aflatoxin BI per kg body weight; B, postmitochondrial supernatant, centrifuged 90 min; C, ribosome pellet resuspendedin 10 mM Tris-HCI (pH 7.1), 0.3 M KC4 and 5 mM MgSOa centrifuged 240 min.
TABLE I TRANSFER RNA CONTRNT OF RIROSOMRS
4s:ss mtio with Ptvnase 1.7 (1) Normal polysomal ribosomes (2) Control ribosomes(propyleneglycol 1.7 + 18 h starvation) 0.7 (3) Starvation monosomes 0.6 (4) Aflatoxin B1 monosomes
4s:ss ratio without Prottase 0.9 0.9 0.7 0.6
Table I, line 1, demonstrates the tRNA content of normal polysomes. When ribosomes were incubated with Pronase the 4 S to 5 S RNA ratio was 1.7; but when ribosomes were extracted without Pronase treatment the 4 S to 5 S RNA ratio decreased to 0.9. Thus, normal ribosomes contain 0.9 molecule of tRNA and/or aminoacyl tRNA and 0.8 molecule of peptidyl tRNA. As seen in Table I, line 2, the ribosomes from control rats have a 4 S to 5 S RNA ratio of 1.7 when incubated with Psonase and a ratio of 0.9 without Pronase treatment. Thus control ribosomes do not differ from normal ribosomes in their tRNA content. Table I, line 3, reveals that monosomes produced by starvation of mice for 72 h contain 0.7 molecule tRNA when incubated with Pronase and without Pronase treatment. This implies that these starvation monosomes contain 0.7 molecule of tRNA and/or aminoacyl tRNA but lack peptidyl tRNAsB7. Similarly, as seen in Table I, line 4, the monosomes produced by aflatoxin A contain 0.6 molecule of tRNA with and without Pronase treatment. This result indicates that the aflatoxin monosomes resemble monosomes produced by starvation and contain 0.6 molecule of tRNA and/or aminoacyl tRNA but no peptidyl tRNA. DISCUssxON
We have shown in agreement with others s-4 that a single injection of aflatoxin Br disaggregates rat liver polysomes into monosomes within 18 h. It has been previously shown that aflatoxin Br inhibits nucleoplasmic RNA polymerase Btr which is presumably responsible for messenger RNA synthesisl*. Inhibition of messenger RNA synthesis could account for the decreased amount of liver polysomes seen witllin 18 h after injection of aflatoxin Bl. These results are consistent with our finding that the monosomes produced by atlatoxin Br dissociated into subunits in buffer containing 0.3 M KCI, indicating that they lack messenger RNAE-‘. We also found that these monosomes contained less than one molecule of tRNA and/or aminoacyl tRNA, but no prptidyl tRNA, similar to the monosomes produced by starvation. The observations that the monosomes produced by aflatoxin Bl are dissociable into subunits and lack peptidyl tRNA suggest that they are runoff ribosomessJ.
347 REFERENCES 1 S. Villa-Tnvino and D. D. Leaver, Effects of the hepatotoxic agents retrorsine and a&toxin BJ on hepatic protein synthesii in the rat, B&hem. J., 109 (1968) 87-91. 2 A. Sarasin and Y. Mottle, In viva elfect of a&toxin L on protein synthesii in rat liver, FEB&’ Lerrers, 29 (1973) 329-332. 3 A. K. Roy, ES&s of aflatoxin BI on polysomal profiles and RNA synthesis in rat Iiver, B&h/m. Biophys. Actu, 169 (1969) 206211. 4 R. S. Pang and 0. N. Wopan, Tie course of alterations of rat liver pofysome protiles induced by aflatoxin &, B&hem. Phtwwcol., 18 (1969) 2357-2361. 5 F, V. Plapp and M. Chiga, Dissociation of ribosomes produced by dimethylnitrosamine and Iasiocarpine in mouse liver, l&&em. Phurmao~., 22 (1973) 1681-1682. 6 F. V. Plapp, L. C. Hayes, L. Tilzer and hi. Chiga, ~~iabiIity and transfer RNA content of rno~ produced by di~hyIni~mi~ and starvation, Narurp, 247 (1974) 311-313. 7 L. L. Tilzer, F. V. Plapp, L. C!. Hayes, and M. ChQa, Certain &aracteristics of monosomes produced by CC%, Biochem, PlwmuMof., (in press). 8 G. N. Dessev and K. Grancharov, Precipitation of RNA, DNA, and nucleoprotein particles from very dilute solutions, Anal. Biochem., 53 (1973) 269-271. 9 K. Oda and W. K. Joklik, Hybridization and sedimentution studies on “early” and “late” vaccinia messenger RNA, J. Mol. Biol., 27 (1964) 395-419. 10 D. Kabat, Direct method of measuring transfer ribonucleic acid binding to polyribosomes and to single ribosomes in ceils of higher organisms, Anal. B&hem., 39 (1971) 228-242. 11 E. 0. Akinrimisi, B. J. Benecke and K. H. Seifart, Inhibition of rat-liver RNA polymerase in vitro by aftatoxin Bl in the presence of a microsomal fraction, Europwn J. Bio&vn., 42 (19741 333-339. 12 M. B. Mathews, Mammalian messenger RNA, in P. N. Campbell and F. Dickens @is.), Es%zyr in B~he/~~is$ry, Academb Press, New York, 9, 1973, pp. 59402.
