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Structural requirements for ochratoxin intoxication
Life Sciences Yol . 11, Part I, pp" 603-508, 1972 . Printed in Great Britain
Pergamon Preee
STRUCTURAL RSQUIRBI~NTS FOR OCHRATOEIN INTOBICATION l Fun Sua Chu, Icksam Noh and Chi C . Chang
Food Research Institute and Department of Food Science University of Wisconsin Madison, Wisconsin 53706
(Received 31 January 1972; in final form 7 April 1972) SU!!lARY
ltte tozicity of several ochratozin derivatives and the apparent dissociation constant of the .phenolic hydrozyl group is these derivatives have been studied . Ochratozin C failed to induce Cozic effect after the pheaolic hydrmcyl group was chemically modified . A direct correlation between the dissociation constant for the phenolic hydroxyl group and the acute tozicity was found . It is suggested that the phenolic group in the dissociated form is necessary for ochratozin iatozication . Ochratozins encompass a group of toxic ieocoumaria derivatives produced .b y several stprage fungi including Asveraillus ochraceue . A . melleus . A . sulvhures and Penicillium viridicatum (1, 2, 3) . toxins varies considerably with their structure .
ltte toxicity of these
Ochrstozin A, 7-carbozy-5-
chloro-g-hydroxy-3, 4-dihydro-3R-methylisocoumsrin licked by its 7-carbozyl group to L-ß-phenylalanine, is the moat potent tozia among this series (4-9) . The methyl and ethyl esters (ochratozin C) of ochratozin A have been found ae tout as the tozin itself (S, 9) ; however, ochratozin B, the dechlorinated toaia, is considered to be nontozic to teat animals (4-7) .
In the present
comm~mication, we wish to present evidence for the role of the phenolic hydroxyl group in the dihydroieocottmarin ring of the tozin on the acute tozicity in animals . 1 This work was eupported by the College of Agricultur{tl and Life Sciences, The University of Wisconsin, b~ a grant from the University of Wisconsin Graduate School (project 120409), and by an institutional grant to the University of Wisconeitr from the American Cancer Society (IN-35R~36) .
503
Vol. 11, No. 10
Taaicitq ad Ochratoa~in
6p4
ERPERIMi tiTAL Preparation of ochratozine . Ochratozin A (OA) 2 and ochratoxin B (OB) were produced by Aspergillue ochraceus in rice (10)and were purified by adsorbs d-5 column chromatography and Sephadex Lfl-20 gel filtration (9) .
The methyl and
ethyl ester (OC) of ochratoain A wets prepared from OA through esterification BF 3 according to the method described by Nesheim (11) .
0-Methylated ochratoxin
C (OM-OC) was prepared from ochratoain C using diazomethane (4, 5) .
The purity
and concentration of all the preparations were verified by TLC, spectrophotometric analyses, ae well as the analysis of phenylalaniae content in the sample after hydrolysis with 6N 1~1 for 48 hrs under vacuum using a Spinco Model 120B amino acid analyzer .
Ochratoain
cx,
the hydrolysed product of ochratoxin A, or
7-carboay-5-chloro-8-hydroay-3, 4-dihydro-3R-methylisocoumarin, was obtained from OA after acid hydrolysis and was further purified by Sephadex LH-20 gel filtration (9) . Spectral analyses and spectrophotometric titratione .
The spectra of vari-
ous ochratoaias were determined by analysis of appropriate solutions in a Beckman DU spectrophotometer with a light path of 1 cm .
For epectrophotometric
titratione, solutions in the range of 3 .0 to 4 .0 z 10 -5M of ochrato :ins in 0 .16 M &C1 were titrated with 0 .1 M NaOH in a Radiometer Automatic Titrator type TTTla with a Radiometer Titragraphy type SBR-2 syringe burette as previously described (19) .
Samples were withdrawn from the titration vessel at appropri-
ate pH values, and the absorbaace measured at 333nm and 380nm .
Ochratoxin C
was titrated in 10.6 methanol . Tozicity determinations .
The acute toxicity of different ochratoxine was
determined by a oral feeding assay method using day-old chicks as previously described (9) . 2The abbreviations used are : OA, ochrato :in A ; OB, ochratoain B ; OC, ochratoain C;
Ocx,
7-carbozy-5-chloro-8-hydroxy-3, 4-dihgdro-3R-methylieocoumarin ; OM-OC,
0-methylated ochratoain C ; BSA, bovine serum albumin .
To~city ad Ochratoaln
Yol. 11, No. 10
505
RESULTS AND DISCUSSION The acute toxicity of OA, OB, OM-OC and 0~ for day-old chicks is given in Table I .
The importance of the phenolic hydroxyl group is ochratoains is dem-
onstrated by the fact that ochratoain C is not tout to test animals after chemical modificâtiôa of the hydroxyl group .
Although lSoore and Truelove
reported that both OA and OCY inhibit mitochondrial respiration in vitro (13), we found that ochratoain
a
was not tout to chicks (9) .
The nontoxic effect
of OCY has been shown in rate (14) and in trout (Sinnhuber, private communication) . TABLE I .
Acute toxicity of ochratoain derivatives on day-old chicks a
Ochratozine
(4tg/chick)
OA
150 200
4/10 9/10
OB
1,000
2/10
OM-OC 0~
Chicks died/chicks tested
500
0/9
1,000
0/8
Body weights of the surviving chicks were the same as controls . No abnormality was observed in the surviving chicks . In order to determine the reactivity of the phenolic hydroxyl group in different ochratoains, the dissociation constant of this group was determined . Except OM-OC in which the phenolic hydroxyl group was blocked by methylation, all other derivatives showed a bathchromic shift with increasing molar absorptivity is basic solutions .
The molar absorptivity of ochratoain derivatives at
333nm and 380nm in acidic and basic solutions is shown in Table II .
To deter-
mine the pK of the phenolic hydroxyl group of dihydroieocoumaria in ochratoains, a epectrophotometric titration was employed which takes advantage of the increase in absorption at 380nm and decrease in absorption at 333nm when the undieaociated phenoayl group dissociates to phenozide ions . titration curves for OB, OC, and Oa. by Chu (12) and by Pitout (15) .
Figure 1 shows
The titration curve for OA was reported
The apparent dissociation constant of the
508
Vol . 11, No . 10
Toadcity ad Ochrataain
TABLE II .
Effect of pH on the absorption maxima of Ochratozin derivatives at
~
above 300 nm
Acid Ochratozins
Baee
H
max (nm)
pH
maz (~)
OA
3 .2
333
6100
10 .0
380
7,650
OB
5 .1
318
6500
10 .0
365
10,400
OCa
3 .1
333
6500
10 .3
380
10,200
oa
6 .6
338
5800
12 .O b
380
5,050
ameasured in 30°G Ethanol bvery unstable at pH's above 12 .0
FIGURE 1 Spectrophotometric titration of ochratoain at 25°(
cX, the fraction of the
phenolic hydrozyl group dissociated, was calculated from either the increase in absorbance at 380nm (OC for Ocx; and 365nm for OB -~-~~) absorbaace at 333mß (OC a~ OcX ; and 318nm for OB method described by Chu (12) .
or the decrease in
-g-p. .a ) according to the
Vol. 11, No. 10
Toa~icity ad Ochrataadn
507
phenolic hydrozyl group in OB, OC, and Oa was found to be 7 .95, 7 .14, and 11 .0, respectively . From the present study, it is readily seen that the tozicity of these derivatives ie closely related to the acid dissociation constant of the phenolic hydroayl group .
Ochratoxia A and OC both with a pR near neutral pH (7 .05
7 .10 for OA, see refs . 12 and 15), were fo~md to be the most potent toxins among the derivatives tested (LD 50 in day-old chicks was 135-166, 216, and 1900 ~g per assay for OiA, OC, and OB, respectively, see refs . 7 and 9 ; ochratosin B was not tonic to duckling and rat, refs . 4 and 5) ; whereas Ocz with an eztremely alkaline pR, was found to be non-tonic .
A direct correlation
between the dissociation constant (k) and LD 50 in day-old chicks was observed in ochratosin A and ochratosin B .
The acid dissociation constant of ochratozin
-8 vs B is ten times smaller thaw OIA (c .a . 10 10 - ~) whereas the toxicity of this toxin ie about ten times less .
If this relationship holds true, the tozicity
of ochratozin CY would be 104 less tonic than OA . The chlorine atom in ochratozin A has been considered to play as important role in ochratosin intoxication (S, 9) because ochratosin B, the dechlorinated derivative, has been shown to be non-toxic .
From the results obtained in the
present study, we feel that the chlorine atom in ochratosin A may play an indirect role .
The chlorine atom may have a direct effect on the dissociation
of the phenolic hydroxyl group in ochratozin A and OC, thus rending it toxic . We have demonstrated recently the interaction of ochratozin A with bovine serum albumin (12) and with several enaymee .
The phenolic hydrozyl group in the toxin
appears to play an important role in these interactions .
In the study of
structure and function relationship of different phenol in biological system, Hansch et al . (16) suggested that both the dissociation constant of phenolic hydrozyl group and hydrophobic interaction were important in the binding between the organic compounds and B3A or mitochoadrial proteins, as well as important for the biological activity .
Although we do not have sufficient data
to deuoastratn hydrophobic effects in the present system, the importance of
508
Toxicity ad Ochratoxin
Vol. 11, No. 10
phenolic group in the toxin for the toxicitq and binding are clear .
It ie
postulated that the tozic effect of ochratoxin arises from the interaction of the phenolic group in the toxin with proteins and enzyme in vivo . The correlation between the dissociation constant of the phenolic hydroxyl group of differont eynthatic dihydroisocoumaria derivatives and the acute tozicity of such derivatives ie presently under study . REFERENCES 1.
R .J . van DER MERWE, P .S . STEYN, L . FOURIE, De B . SCOTT, and J .J . THERON, Nature 205 . 1112-1113 (1965) .
2.
M . LAI, G . SEMENIUR, end C .W . HESSELTINE, Phvtopatholostv . 58, 1056 (1968) (Abetr .)
3.
W . van WALBEEK, P .M . SCOTT, J . HARTWIG, and J .W . LAFIRENCE, Can . J . Microbiol . 1281-1285 (1969) .
4.
K .J . van DER MER4TE, P .S . STSYN, and L . FOURIE, J . Chew. Soc . 7083-7088 (1965) . P .S . STEYN, and C .W . HOLZAPFEL, Tetrahedron ~,3_, 4449-4461 (1967)
5. 6:
L F .H . PURCHASE, and W . NEL, In "Biochemietr~ of some foodborne microbial tozine ," R.I . Mateles and G .N . Wogan, ed . MIT Press, Cambridge, Mass . 153-156 (1967) .
7.
L C . PECRHAit, B . DOURIIK, JR ., and O . H . JONES, JR ., Applied Microbiol . 21, 492-494 (1971) .
8.
P .S . STEYN, a~ C .W . HOLZAPFEL, J . So . Africa Chem . Inet . 20, 186-189 (1967) .
9.
F .S . CHU, and C . C . CHANG, J . Am. Official Analvt . Chem . =,1032-1034 (1971) .
10 .
H . TRENR, M. BUTZ, and F .S . CHU, Appl . Microbiol . 21, 1032-1035 (1971),
11 .
S . NESHEIM, J . Am . Official Analvt . Chem. ~2, 975-979 (1969) .
12 .
F .S . CHQ, Arch . Biochem. Biophys . 147 . 359-36 6 (1971) .
13 .
J .H . MOORE, aad B . TRUELOVE, S
14 .
P . STILL, A .W . MACRLIN, W .E . RIBELIN, and E . B . SMALLSY, Nature 234 . 563-564 (1971) .
15 .
M .J . PITOUT, To:icol . and ADpl . Pharmacol . 13, 299-306 (1968) .
16 .
C . BANSCH, R . &IEHS, and G .L . LlWRENCE, J . Am . Chem . Soc . ~, 5770-5773 (1965) .
e ; 16~, 1102-1103 (1970) .
Pergamon Preee
STRUCTURAL RSQUIRBI~NTS FOR OCHRATOEIN INTOBICATION l Fun Sua Chu, Icksam Noh and Chi C . Chang
Food Research Institute and Department of Food Science University of Wisconsin Madison, Wisconsin 53706
(Received 31 January 1972; in final form 7 April 1972) SU!!lARY
ltte tozicity of several ochratozin derivatives and the apparent dissociation constant of the .phenolic hydrozyl group is these derivatives have been studied . Ochratozin C failed to induce Cozic effect after the pheaolic hydrmcyl group was chemically modified . A direct correlation between the dissociation constant for the phenolic hydroxyl group and the acute tozicity was found . It is suggested that the phenolic group in the dissociated form is necessary for ochratozin iatozication . Ochratozins encompass a group of toxic ieocoumaria derivatives produced .b y several stprage fungi including Asveraillus ochraceue . A . melleus . A . sulvhures and Penicillium viridicatum (1, 2, 3) . toxins varies considerably with their structure .
ltte toxicity of these
Ochrstozin A, 7-carbozy-5-
chloro-g-hydroxy-3, 4-dihydro-3R-methylisocoumsrin licked by its 7-carbozyl group to L-ß-phenylalanine, is the moat potent tozia among this series (4-9) . The methyl and ethyl esters (ochratozin C) of ochratozin A have been found ae tout as the tozin itself (S, 9) ; however, ochratozin B, the dechlorinated toaia, is considered to be nontozic to teat animals (4-7) .
In the present
comm~mication, we wish to present evidence for the role of the phenolic hydroxyl group in the dihydroieocottmarin ring of the tozin on the acute tozicity in animals . 1 This work was eupported by the College of Agricultur{tl and Life Sciences, The University of Wisconsin, b~ a grant from the University of Wisconsin Graduate School (project 120409), and by an institutional grant to the University of Wisconeitr from the American Cancer Society (IN-35R~36) .
503
Vol. 11, No. 10
Taaicitq ad Ochratoa~in
6p4
ERPERIMi tiTAL Preparation of ochratozine . Ochratozin A (OA) 2 and ochratoxin B (OB) were produced by Aspergillue ochraceus in rice (10)and were purified by adsorbs d-5 column chromatography and Sephadex Lfl-20 gel filtration (9) .
The methyl and
ethyl ester (OC) of ochratoain A wets prepared from OA through esterification BF 3 according to the method described by Nesheim (11) .
0-Methylated ochratoxin
C (OM-OC) was prepared from ochratoain C using diazomethane (4, 5) .
The purity
and concentration of all the preparations were verified by TLC, spectrophotometric analyses, ae well as the analysis of phenylalaniae content in the sample after hydrolysis with 6N 1~1 for 48 hrs under vacuum using a Spinco Model 120B amino acid analyzer .
Ochratoain
cx,
the hydrolysed product of ochratoxin A, or
7-carboay-5-chloro-8-hydroay-3, 4-dihydro-3R-methylisocoumarin, was obtained from OA after acid hydrolysis and was further purified by Sephadex LH-20 gel filtration (9) . Spectral analyses and spectrophotometric titratione .
The spectra of vari-
ous ochratoaias were determined by analysis of appropriate solutions in a Beckman DU spectrophotometer with a light path of 1 cm .
For epectrophotometric
titratione, solutions in the range of 3 .0 to 4 .0 z 10 -5M of ochrato :ins in 0 .16 M &C1 were titrated with 0 .1 M NaOH in a Radiometer Automatic Titrator type TTTla with a Radiometer Titragraphy type SBR-2 syringe burette as previously described (19) .
Samples were withdrawn from the titration vessel at appropri-
ate pH values, and the absorbaace measured at 333nm and 380nm .
Ochratoxin C
was titrated in 10.6 methanol . Tozicity determinations .
The acute toxicity of different ochratoxine was
determined by a oral feeding assay method using day-old chicks as previously described (9) . 2The abbreviations used are : OA, ochrato :in A ; OB, ochratoain B ; OC, ochratoain C;
Ocx,
7-carbozy-5-chloro-8-hydroxy-3, 4-dihgdro-3R-methylieocoumarin ; OM-OC,
0-methylated ochratoain C ; BSA, bovine serum albumin .
To~city ad Ochratoaln
Yol. 11, No. 10
505
RESULTS AND DISCUSSION The acute toxicity of OA, OB, OM-OC and 0~ for day-old chicks is given in Table I .
The importance of the phenolic hydroxyl group is ochratoains is dem-
onstrated by the fact that ochratoain C is not tout to test animals after chemical modificâtiôa of the hydroxyl group .
Although lSoore and Truelove
reported that both OA and OCY inhibit mitochondrial respiration in vitro (13), we found that ochratoain
a
was not tout to chicks (9) .
The nontoxic effect
of OCY has been shown in rate (14) and in trout (Sinnhuber, private communication) . TABLE I .
Acute toxicity of ochratoain derivatives on day-old chicks a
Ochratozine
(4tg/chick)
OA
150 200
4/10 9/10
OB
1,000
2/10
OM-OC 0~
Chicks died/chicks tested
500
0/9
1,000
0/8
Body weights of the surviving chicks were the same as controls . No abnormality was observed in the surviving chicks . In order to determine the reactivity of the phenolic hydroxyl group in different ochratoains, the dissociation constant of this group was determined . Except OM-OC in which the phenolic hydroxyl group was blocked by methylation, all other derivatives showed a bathchromic shift with increasing molar absorptivity is basic solutions .
The molar absorptivity of ochratoain derivatives at
333nm and 380nm in acidic and basic solutions is shown in Table II .
To deter-
mine the pK of the phenolic hydroxyl group of dihydroieocoumaria in ochratoains, a epectrophotometric titration was employed which takes advantage of the increase in absorption at 380nm and decrease in absorption at 333nm when the undieaociated phenoayl group dissociates to phenozide ions . titration curves for OB, OC, and Oa. by Chu (12) and by Pitout (15) .
Figure 1 shows
The titration curve for OA was reported
The apparent dissociation constant of the
508
Vol . 11, No . 10
Toadcity ad Ochrataain
TABLE II .
Effect of pH on the absorption maxima of Ochratozin derivatives at
~
above 300 nm
Acid Ochratozins
Baee
H
max (nm)
pH
maz (~)
OA
3 .2
333
6100
10 .0
380
7,650
OB
5 .1
318
6500
10 .0
365
10,400
OCa
3 .1
333
6500
10 .3
380
10,200
oa
6 .6
338
5800
12 .O b
380
5,050
ameasured in 30°G Ethanol bvery unstable at pH's above 12 .0
FIGURE 1 Spectrophotometric titration of ochratoain at 25°(
cX, the fraction of the
phenolic hydrozyl group dissociated, was calculated from either the increase in absorbance at 380nm (OC for Ocx; and 365nm for OB -~-~~) absorbaace at 333mß (OC a~ OcX ; and 318nm for OB method described by Chu (12) .
or the decrease in
-g-p. .a ) according to the
Vol. 11, No. 10
Toa~icity ad Ochrataadn
507
phenolic hydrozyl group in OB, OC, and Oa was found to be 7 .95, 7 .14, and 11 .0, respectively . From the present study, it is readily seen that the tozicity of these derivatives ie closely related to the acid dissociation constant of the phenolic hydroayl group .
Ochratoxia A and OC both with a pR near neutral pH (7 .05
7 .10 for OA, see refs . 12 and 15), were fo~md to be the most potent toxins among the derivatives tested (LD 50 in day-old chicks was 135-166, 216, and 1900 ~g per assay for OiA, OC, and OB, respectively, see refs . 7 and 9 ; ochratosin B was not tonic to duckling and rat, refs . 4 and 5) ; whereas Ocz with an eztremely alkaline pR, was found to be non-tonic .
A direct correlation
between the dissociation constant (k) and LD 50 in day-old chicks was observed in ochratosin A and ochratosin B .
The acid dissociation constant of ochratozin
-8 vs B is ten times smaller thaw OIA (c .a . 10 10 - ~) whereas the toxicity of this toxin ie about ten times less .
If this relationship holds true, the tozicity
of ochratozin CY would be 104 less tonic than OA . The chlorine atom in ochratozin A has been considered to play as important role in ochratosin intoxication (S, 9) because ochratosin B, the dechlorinated derivative, has been shown to be non-toxic .
From the results obtained in the
present study, we feel that the chlorine atom in ochratosin A may play an indirect role .
The chlorine atom may have a direct effect on the dissociation
of the phenolic hydroxyl group in ochratozin A and OC, thus rending it toxic . We have demonstrated recently the interaction of ochratozin A with bovine serum albumin (12) and with several enaymee .
The phenolic hydrozyl group in the toxin
appears to play an important role in these interactions .
In the study of
structure and function relationship of different phenol in biological system, Hansch et al . (16) suggested that both the dissociation constant of phenolic hydrozyl group and hydrophobic interaction were important in the binding between the organic compounds and B3A or mitochoadrial proteins, as well as important for the biological activity .
Although we do not have sufficient data
to deuoastratn hydrophobic effects in the present system, the importance of
508
Toxicity ad Ochratoxin
Vol. 11, No. 10
phenolic group in the toxin for the toxicitq and binding are clear .
It ie
postulated that the tozic effect of ochratoxin arises from the interaction of the phenolic group in the toxin with proteins and enzyme in vivo . The correlation between the dissociation constant of the phenolic hydroxyl group of differont eynthatic dihydroisocoumaria derivatives and the acute tozicity of such derivatives ie presently under study . REFERENCES 1.
R .J . van DER MERWE, P .S . STEYN, L . FOURIE, De B . SCOTT, and J .J . THERON, Nature 205 . 1112-1113 (1965) .
2.
M . LAI, G . SEMENIUR, end C .W . HESSELTINE, Phvtopatholostv . 58, 1056 (1968) (Abetr .)
3.
W . van WALBEEK, P .M . SCOTT, J . HARTWIG, and J .W . LAFIRENCE, Can . J . Microbiol . 1281-1285 (1969) .
4.
K .J . van DER MER4TE, P .S . STSYN, and L . FOURIE, J . Chew. Soc . 7083-7088 (1965) . P .S . STEYN, and C .W . HOLZAPFEL, Tetrahedron ~,3_, 4449-4461 (1967)
5. 6:
L F .H . PURCHASE, and W . NEL, In "Biochemietr~ of some foodborne microbial tozine ," R.I . Mateles and G .N . Wogan, ed . MIT Press, Cambridge, Mass . 153-156 (1967) .
7.
L C . PECRHAit, B . DOURIIK, JR ., and O . H . JONES, JR ., Applied Microbiol . 21, 492-494 (1971) .
8.
P .S . STEYN, a~ C .W . HOLZAPFEL, J . So . Africa Chem . Inet . 20, 186-189 (1967) .
9.
F .S . CHU, and C . C . CHANG, J . Am. Official Analvt . Chem . =,1032-1034 (1971) .
10 .
H . TRENR, M. BUTZ, and F .S . CHU, Appl . Microbiol . 21, 1032-1035 (1971),
11 .
S . NESHEIM, J . Am . Official Analvt . Chem. ~2, 975-979 (1969) .
12 .
F .S . CHQ, Arch . Biochem. Biophys . 147 . 359-36 6 (1971) .
13 .
J .H . MOORE, aad B . TRUELOVE, S
14 .
P . STILL, A .W . MACRLIN, W .E . RIBELIN, and E . B . SMALLSY, Nature 234 . 563-564 (1971) .
15 .
M .J . PITOUT, To:icol . and ADpl . Pharmacol . 13, 299-306 (1968) .
16 .
C . BANSCH, R . &IEHS, and G .L . LlWRENCE, J . Am . Chem . Soc . ~, 5770-5773 (1965) .
e ; 16~, 1102-1103 (1970) .