Fd Chem. Toxic. Vol. 30, No. 4, pp. 277-279, 1992 Printed in Great Britain. All rights reserved
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
INITIAL STUDIES ON THE TOXICOKINETICS OF F U M O N I S I N BI IN RATS G. S. SHEPHARD*,P. G. THIEL and E. W. SYDEI~,AM Programme on Mycotoxins and Experimental Carcinogenesis, South African Medical Research Council, PO Box 19070, Tygerberg 7505, South Africa (Accepted 18 December 1991) Abstract--Fumonisin B~ (FB,), the major compound in the fumonisin group of secondary metabolites of Fusarium moniliforme Sheldon, is associated with some human and animal diseases. After intraperitoneal dosing to rats (7.5 mg/kg), FB, was rapidly absorbed and reached a maximum concentration in plasma within 20 min after injection. Thereafter, it underwent rapid removal from plasma, displaying a mono-exponential elimination phase that fitted a one-compartment model with a half-life of 18 min. Collection of 24- and 48-hr urine samples indicated that only 16% of the applied dose was eliminated unmetabolized in urine, all within the first 24-hr period following dosing. In contrast to this, a similar dose of FB I given by gavage resulted in the recovery of only 0.4% of the FBI in urine.
INTRODUCTION The fumonisins are a group of recently described, structurally similar, secondary metabolites of the fungus Fusarium moniliforme Sheldon, a common contaminant of corn worldwide (Bezuidenhout et al., 1988; Cawood et al., 1991; Gelderblom et al., 1988). Of these compounds, fumonisin BI (FBI) has been found to be the major product both in corn culture and in naturally contaminated feed and foodstuffs (Ross et al., 1991; Shephard et al., 1990; Sydenham et al., 1991; Thiel et al., 1991b,c). Thus most of the toxicological data published to date have concerned the effects of FB] on different animal species. FB~ was first isolated from fungal cultures of F. moniliforme MRC 826 (Gelderblom et al., 1988). It was shown to be the causative agent of leukoencephalomalacia in horses by the induction of the disease first by iv injection of the pure compound at repeated doses of 0.125 mg/kg body weight (Marasas et al., 1988) and subsequently by daily oral administration at levels between 1 and 4 mg/kg body weight (Kellerman et al., 1990). It has since also been shown that iv injection of pure FB] in swine at levels of 0.4 mg/kg can cause pulmonary oedema (Harrison et al., 1990). FB~ has been shown to be hepatocarcinogenic and hepatotoxic when fed to rats at a level of 50 mg/kg diet over periods up to 26 months (Gelderblom et al., 1991). Although not yet definitely linked to any diseases in humans, FBm has been associated with an increased risk of oesophageal cancer in the inhabitants of rural areas in Transkei, southern Africa (Rheeder et al., 1992; Sydenham et al., 1990). *To whom all correspondence should be addressed. Abbreviations: FB, •fumonisin B~; SAXffistrong anion exchange. rcr 3o/4-a
In view of the toxicological effects of FB~, this study was undertaken to determine some of the toxicokinetic parameters of FBm in rats. In particular, the plasma clearance and urinary excretion of FBm were investigated following ip injection of the toxin; this was compared with the urinary excretion after administration of the toxin by gavage. MATERIALS AND METHODS
Animals and chemicals Male BD IX rats were bred at the Research Institute for Nutritional Diseases, Tygerberg, South Africa. At the time of the experiment they were approx. 6 wk old and weighed approx. 150 g. FBI was isolated from F. moniliforme MRC 826 fungal cultures at the Research Institute for Nutritional Diseases as previously described (Cawood et al., 1991; Gelderblom et al., 1988). All other chemicals were of analytical grade. Experimental procedures (i) Clearance of FB l from plasma after ip injection. Fourteen rats were confined in individual cages. At the start of the experiment they were all given, at recorded times, an ip injection of FB~ in 0.9% saline at a dose of 7.5 mg/kg body weight, a dose of similar magnitude to those used in previous toxicological experiments (Thiel et al., 1991a). Rats were killed in pairs after time periods of 6, 10, 20, 30, 60, 120 and 180 rnin. Blood was obtained by exsanguination of anaesthetized (by ip injection of sodium pentobarbitone solution) rats through the abdominal aorta using a needle and syringe. The blood was transferred to blood collection tubes containing tripotassium ethylenediaminetetra-acetate as anticoagulant. Plasma was obtained by centrifugation at 1200g for lOmin at 4°C. 277
278
G.S. SHEPHAROet al. 10-
(i0 Excretion of FB 1 after ip injection. Two groups of four rats each were confined in metabolic cages with free access to feed and water. At the start of the experiment each rat was given an ip injection of FB~ in 0.9% saline at a dose of 7.5 mg/kg body weight. Urine was collected for 24 hr in the one group and for 48 hr in the other group. At the end of these periods, the rats were killed by exsanguination and plasma was obtained as in part (i). (iii) Excretion of FB I after oral dosing. The above experiment in part (ii) was repeated, except that the rats were dosed by gavage with an aqueous solution of FBI at 7.5 mg/kg body weight, the same dosage level as the ip injection.
f~
E
:3.
v C 0 ".if, t-
4
O
C 0
O
2 LI.
o
I
I
20
40
Analytical methods FB~ was determined in plasma and urine by reversed-phase HPLC of its o-phthaldialdehyde derivative following sample clean-up using BondElut (Analytichem International, Harbor City, CA, USA) strong anion exchange (SAX) solid phase extraction cartridges (3 ml capacity, 500 mg sorbent) (Shephard et al., 1992). Briefly, the SAX cartridges were conditioned before the application of samples with 5 ml methanol and 5 ml methanol-water (3:1, v/v). Plasma samples (500/~1) were prepared for analysis by addition of 2.5 ml methanol in order to precipitate plasma proteins. After centrifugation, 2 ml supernatant (pH approx. 8.7) was applied to the clean-up cartridge. Urine samples (250/zl; pH approx. 6) were diluted with 750 #1 water and 3 ml methanol before clean-up. In all determinations, the SAX cartridge was washed after sample application with 5 ml methanol-water (3:1, v/v) followed by 5 ml methanol, and FB~ was eluted with 10 ml 5% acetic acid in methanol. For all operations, the flow rate through the SAX cartridge was approx. 1.0-1.5 ml/ min. The eluate was dried and FB~ was quantified as previously described (Shephard et al., 1992).
60
80
100
120
140
T i m e (rain)
Fig 1. Variation of plasma concentration of FB~ with time following ip dosage of the toxin. Each point represents the mean of determinations in two rats. of FB l is rapid and after 3 hr only trace levels (less than 100 ng/g) were found. The data in Fig. 1 were redrawn to yield a semilogarithmic plot of plasma concentration against time for those points measured in the elimination phase (Fig. 2). The linear nature of this plot indicates first-order kinetics with a single elimination phase (mono-exponentiai) and hence the applicability of a one-compartment model to describe the toxicokinetics of FB~ in rats. Such a model implies rapid equilibrium of FB~ between body tissues. A straight-line linear regression fit to the relevant time points gave a slope from which was calculated the elimination rate constant of 0.039 min- t. Hence the value of the half-life for the disappearance of FB~ from rat plasma (Tl/~) was calculated as 18 rain. The renal excretion of FB~ following the ip administration of toxin was determined in two groups of four rats each. The first group, killed after 24 hr, gave a total unmetabolized FB~ excretion of 16.6% of the administered dose; the second group, killed after
RESULTS AND DISCUSSION
The rat plasma concentrations of FB l were determined at varying time intervals after the ip injection of FB~. The nature of this time dependence is shown in Fig. 1 for the first 2 hr after injection. Each point represents the mean of the levels determined in each of two rats. The rapid absorption of FBI following the ip dose is clearly shown by the rapid rise in FB~ levels such that the maximum concentration (Cmax) of 8.6 #g/ml occurred within 20 min (TmJ. At this time point, assuming a plasma volume of 5.3 ml for a 150-g rat (Waynforth, 1980), the maximum concentration represents the presence in plasma of approx. 4% of the total dose or an apparent volume of distribution of the order of 130 ml. After 20 min the sharp decline in plasma levels of FB1 represents the elimination phase for the toxin, the absorption and distribution phases having clearly been completed within the first 20 min. The clearance
10 - -
x\ x
tO e,.
0 C 0
0
0.1
I
I
I
I
20
40
60
80
I
I
I
100 120 140
Time (rain)
Fig. 2. Semi-logarithmic plot of the elimination phase of FB~
from plasma after ip administration of the toxin.
Toxicokinetic studies on fumonisin B~ in rats 48 hr gave a very similar excretion of 15.3%. Hence, following ip injection, approx. 16% of the administered dose of FB~ appeared unchanged in urine within 24 hr and no further urinary excretion of the toxin itself occurred. The limitation of the excretion of FB~ to the first 24 hr after injection is consistent with the rapid elimination of the toxin from plasma. In order to test the effect of the route of exposure to FB~ on its urinary excretion, two groups of four rats each were again confined in metabolic cages and dosed by gavage with the same FB~ dose used for the ip groups. Analysis of urine indicated that less than 0.4% of the applied dose appeared in the urine, all within the first 24 hr of dosing; hence the bulk of the dose remained undetected and its metabolic fate unknown. Further work to determine the metabolic fate of FB~ and to identify any unknown metabolites will require the use of radiolabelled FB~. In conclusion, this study has shown that following ip dosing of FB~, both the uptake of the toxin into the bloodstream and the elimination of the toxin are rapid, as indicated by a Tm,x of less than 20 rain and by a half-life in plasma of 18 min. However, of the total dose, only 16% could be accounted for as unmetabolized toxin in urine. The fate of the remaining 84% (and of nearly the total dose when given orally) is unknown and remains the subject of further investigation.
Acknowledgements--The authors wish to thank H. J. B. Joubert and P. W. De Lange for technical assistance in the handling of laboratory animals and W. C. A. Gelderblom and M. E. Cawood for the provision of FB I . REFERENCES
Bezuidenhout S. C., Gelderblom W. C. A., Gorst-Allman C. P., Horak R. M., Marasas W. F. O., Spiteller G. and Vleggaar R. (1988) Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme. Journal of the Chemical Society, Chemical Communications 743-745. Cawood M. E., Gelderhlom W. C. A., Vleggaar R., Behrend Y., Thiel P. G. and Marasas W. F. O. (1991) Isolation of the fumonisin mycotoxins--a quantitative approach. Journal of Agricultural and Food Chemistry 39, 1958-1962. Gelderblom W. C. A., Jaskiewickz K., Marasas W. F. O., Thiel P. G., Horak R. M., Vleggaar R. and Kriek N. P. J. (1988) Fumonisins--novel mycotoxins with cancerpromoting activity produced by Fusarium moniliforme. Applied and Environmental Microbiology 54, 1806-1811. Gelderblom W. C. A., Kriek N. P. J., Marasas W. F. O. and Thiel P. G. (1991) Toxicity and carcinogenicity of the
279
Fusarium moniliforme metabolite, fumonisin Bi, in rats. Carcinogenesis 12, 1247-1251. Harrison L. R., Colvin B. M., Greene J. T., Newman L. E. and Cole J. R. (1990) Pulmonary edema and hydrothorax in swine produced by fumonisin Bt, a toxic metabolite of Fusarium moniliforme. Journal of Veterinary Diagnostic Investigation 2, 217-221. Kellerman T. S., Marasas W. F. O., Thiel P. G., Gelderblom W. C. A., Cawood M. and Coetzer J. A. W. (1990) Leukoencephalomalacia in two horses induced by oral dosing of fumonisin Bt . Onderstepoort Journal of Veterinary Research 57, 269-275. Marasas W. F. O., Kellerman T. S., Gelderblom W. C. A., Coetzer J. A. W., Thiel P. G. and Van der Lugt J. J. (1988) Leukoencephalomalacia in a horse induced by fumonisin B~, isolated from Fusarium moniliforme. Onderstepoort Journal of Veterinary Research 55, 197-203. Rheeder J. P., Marasas W. F. O., Thiel P. G., Sydenham E. W., Shephard G. S. and Van Schalkwyk D. J. (1992) Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei. Phytopathology 82, 353-357. Ross P. F., Rice L. G., Plattner R. D., Osweiler G. D., Wilson T. M., Owens D. L., Nelson H. A. and Richard J. L. (1991) Concentrations of fumonisin BI in feeds associated with animal health problems. Mycopathologia 114, 129-135. Shephard G. S., Sydenham E. W., Thiel P. G. and Gelderblom W. C. A. (1990) Quantitative determination of fumonisins Bt and B 2 by high-performance liquid chromatography with fluorescence detection. Journal of Liquid Chromatography 13, 2077-2087. Shephard G. S., Thiel P. G. and Sydenham E. W. (1992) Determination of fumonisin Bt in plasma and urine by high-performance liquid chromatography. Journal of Chromatography 574, 299-304. Sydenham E. W., Shephard G. S., Thiel P. G., Marasas W.F.O. and Stockenstr6m S. (1991) Fumonisin contamination of commercial corn-based human foodstuffs. Journal of Agricultural and Food Chemistry 39, 2014-2018. Sydenham E. W., Thiel P. G., Marasas W. F. O., Shephard G. S., Van Schalkwyk D. J. and Koch K. R. (1990) Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, southern Africa. Journal of Agricultural and Food Chemistry 38, 1900-1903. Thiel P. G., Marasas W. F. O., Sydenham E. W., Shephard G. S. and Gelderblom W. C. A. (1991a) The implications of naturally occurring levels of fumonisins in corn for human and animal health. Mycopathologia. In press. Thiel P. G., Marasas W. F. O., Sydenham E. W., Shephard G. S., Gelderblom W. C. A. and NieuwenhuisJ. J. (1991b) Survey of fumonisin production by Fusarium species. Applied Environmental Microbiology 57, 1089-1093. Thiel P. G., Shephard G. S., Sydenham E. W., Marasas W. F. O., Nelson P. E. and Wilson T. M. (1991c) Levels of fumonisins Bt and B2 in feeds associated with confirmed cases of equine leukoencephalomalacia. Journal of Agricultural and Food Chemistry 39, 109-111. Waynforth H. B. (1980) Experimental and Surgical Techniques in the Rat. p. 240. Academic Press, London.
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
INITIAL STUDIES ON THE TOXICOKINETICS OF F U M O N I S I N BI IN RATS G. S. SHEPHARD*,P. G. THIEL and E. W. SYDEI~,AM Programme on Mycotoxins and Experimental Carcinogenesis, South African Medical Research Council, PO Box 19070, Tygerberg 7505, South Africa (Accepted 18 December 1991) Abstract--Fumonisin B~ (FB,), the major compound in the fumonisin group of secondary metabolites of Fusarium moniliforme Sheldon, is associated with some human and animal diseases. After intraperitoneal dosing to rats (7.5 mg/kg), FB, was rapidly absorbed and reached a maximum concentration in plasma within 20 min after injection. Thereafter, it underwent rapid removal from plasma, displaying a mono-exponential elimination phase that fitted a one-compartment model with a half-life of 18 min. Collection of 24- and 48-hr urine samples indicated that only 16% of the applied dose was eliminated unmetabolized in urine, all within the first 24-hr period following dosing. In contrast to this, a similar dose of FB I given by gavage resulted in the recovery of only 0.4% of the FBI in urine.
INTRODUCTION The fumonisins are a group of recently described, structurally similar, secondary metabolites of the fungus Fusarium moniliforme Sheldon, a common contaminant of corn worldwide (Bezuidenhout et al., 1988; Cawood et al., 1991; Gelderblom et al., 1988). Of these compounds, fumonisin BI (FBI) has been found to be the major product both in corn culture and in naturally contaminated feed and foodstuffs (Ross et al., 1991; Shephard et al., 1990; Sydenham et al., 1991; Thiel et al., 1991b,c). Thus most of the toxicological data published to date have concerned the effects of FB] on different animal species. FB~ was first isolated from fungal cultures of F. moniliforme MRC 826 (Gelderblom et al., 1988). It was shown to be the causative agent of leukoencephalomalacia in horses by the induction of the disease first by iv injection of the pure compound at repeated doses of 0.125 mg/kg body weight (Marasas et al., 1988) and subsequently by daily oral administration at levels between 1 and 4 mg/kg body weight (Kellerman et al., 1990). It has since also been shown that iv injection of pure FB] in swine at levels of 0.4 mg/kg can cause pulmonary oedema (Harrison et al., 1990). FB~ has been shown to be hepatocarcinogenic and hepatotoxic when fed to rats at a level of 50 mg/kg diet over periods up to 26 months (Gelderblom et al., 1991). Although not yet definitely linked to any diseases in humans, FBm has been associated with an increased risk of oesophageal cancer in the inhabitants of rural areas in Transkei, southern Africa (Rheeder et al., 1992; Sydenham et al., 1990). *To whom all correspondence should be addressed. Abbreviations: FB, •fumonisin B~; SAXffistrong anion exchange. rcr 3o/4-a
In view of the toxicological effects of FB~, this study was undertaken to determine some of the toxicokinetic parameters of FBm in rats. In particular, the plasma clearance and urinary excretion of FBm were investigated following ip injection of the toxin; this was compared with the urinary excretion after administration of the toxin by gavage. MATERIALS AND METHODS
Animals and chemicals Male BD IX rats were bred at the Research Institute for Nutritional Diseases, Tygerberg, South Africa. At the time of the experiment they were approx. 6 wk old and weighed approx. 150 g. FBI was isolated from F. moniliforme MRC 826 fungal cultures at the Research Institute for Nutritional Diseases as previously described (Cawood et al., 1991; Gelderblom et al., 1988). All other chemicals were of analytical grade. Experimental procedures (i) Clearance of FB l from plasma after ip injection. Fourteen rats were confined in individual cages. At the start of the experiment they were all given, at recorded times, an ip injection of FB~ in 0.9% saline at a dose of 7.5 mg/kg body weight, a dose of similar magnitude to those used in previous toxicological experiments (Thiel et al., 1991a). Rats were killed in pairs after time periods of 6, 10, 20, 30, 60, 120 and 180 rnin. Blood was obtained by exsanguination of anaesthetized (by ip injection of sodium pentobarbitone solution) rats through the abdominal aorta using a needle and syringe. The blood was transferred to blood collection tubes containing tripotassium ethylenediaminetetra-acetate as anticoagulant. Plasma was obtained by centrifugation at 1200g for lOmin at 4°C. 277
278
G.S. SHEPHAROet al. 10-
(i0 Excretion of FB 1 after ip injection. Two groups of four rats each were confined in metabolic cages with free access to feed and water. At the start of the experiment each rat was given an ip injection of FB~ in 0.9% saline at a dose of 7.5 mg/kg body weight. Urine was collected for 24 hr in the one group and for 48 hr in the other group. At the end of these periods, the rats were killed by exsanguination and plasma was obtained as in part (i). (iii) Excretion of FB I after oral dosing. The above experiment in part (ii) was repeated, except that the rats were dosed by gavage with an aqueous solution of FBI at 7.5 mg/kg body weight, the same dosage level as the ip injection.
f~
E
:3.
v C 0 ".if, t-
4
O
C 0
O
2 LI.
o
I
I
20
40
Analytical methods FB~ was determined in plasma and urine by reversed-phase HPLC of its o-phthaldialdehyde derivative following sample clean-up using BondElut (Analytichem International, Harbor City, CA, USA) strong anion exchange (SAX) solid phase extraction cartridges (3 ml capacity, 500 mg sorbent) (Shephard et al., 1992). Briefly, the SAX cartridges were conditioned before the application of samples with 5 ml methanol and 5 ml methanol-water (3:1, v/v). Plasma samples (500/~1) were prepared for analysis by addition of 2.5 ml methanol in order to precipitate plasma proteins. After centrifugation, 2 ml supernatant (pH approx. 8.7) was applied to the clean-up cartridge. Urine samples (250/zl; pH approx. 6) were diluted with 750 #1 water and 3 ml methanol before clean-up. In all determinations, the SAX cartridge was washed after sample application with 5 ml methanol-water (3:1, v/v) followed by 5 ml methanol, and FB~ was eluted with 10 ml 5% acetic acid in methanol. For all operations, the flow rate through the SAX cartridge was approx. 1.0-1.5 ml/ min. The eluate was dried and FB~ was quantified as previously described (Shephard et al., 1992).
60
80
100
120
140
T i m e (rain)
Fig 1. Variation of plasma concentration of FB~ with time following ip dosage of the toxin. Each point represents the mean of determinations in two rats. of FB l is rapid and after 3 hr only trace levels (less than 100 ng/g) were found. The data in Fig. 1 were redrawn to yield a semilogarithmic plot of plasma concentration against time for those points measured in the elimination phase (Fig. 2). The linear nature of this plot indicates first-order kinetics with a single elimination phase (mono-exponentiai) and hence the applicability of a one-compartment model to describe the toxicokinetics of FB~ in rats. Such a model implies rapid equilibrium of FB~ between body tissues. A straight-line linear regression fit to the relevant time points gave a slope from which was calculated the elimination rate constant of 0.039 min- t. Hence the value of the half-life for the disappearance of FB~ from rat plasma (Tl/~) was calculated as 18 rain. The renal excretion of FB~ following the ip administration of toxin was determined in two groups of four rats each. The first group, killed after 24 hr, gave a total unmetabolized FB~ excretion of 16.6% of the administered dose; the second group, killed after
RESULTS AND DISCUSSION
The rat plasma concentrations of FB l were determined at varying time intervals after the ip injection of FB~. The nature of this time dependence is shown in Fig. 1 for the first 2 hr after injection. Each point represents the mean of the levels determined in each of two rats. The rapid absorption of FBI following the ip dose is clearly shown by the rapid rise in FB~ levels such that the maximum concentration (Cmax) of 8.6 #g/ml occurred within 20 min (TmJ. At this time point, assuming a plasma volume of 5.3 ml for a 150-g rat (Waynforth, 1980), the maximum concentration represents the presence in plasma of approx. 4% of the total dose or an apparent volume of distribution of the order of 130 ml. After 20 min the sharp decline in plasma levels of FB1 represents the elimination phase for the toxin, the absorption and distribution phases having clearly been completed within the first 20 min. The clearance
10 - -
x\ x
tO e,.
0 C 0
0
0.1
I
I
I
I
20
40
60
80
I
I
I
100 120 140
Time (rain)
Fig. 2. Semi-logarithmic plot of the elimination phase of FB~
from plasma after ip administration of the toxin.
Toxicokinetic studies on fumonisin B~ in rats 48 hr gave a very similar excretion of 15.3%. Hence, following ip injection, approx. 16% of the administered dose of FB~ appeared unchanged in urine within 24 hr and no further urinary excretion of the toxin itself occurred. The limitation of the excretion of FB~ to the first 24 hr after injection is consistent with the rapid elimination of the toxin from plasma. In order to test the effect of the route of exposure to FB~ on its urinary excretion, two groups of four rats each were again confined in metabolic cages and dosed by gavage with the same FB~ dose used for the ip groups. Analysis of urine indicated that less than 0.4% of the applied dose appeared in the urine, all within the first 24 hr of dosing; hence the bulk of the dose remained undetected and its metabolic fate unknown. Further work to determine the metabolic fate of FB~ and to identify any unknown metabolites will require the use of radiolabelled FB~. In conclusion, this study has shown that following ip dosing of FB~, both the uptake of the toxin into the bloodstream and the elimination of the toxin are rapid, as indicated by a Tm,x of less than 20 rain and by a half-life in plasma of 18 min. However, of the total dose, only 16% could be accounted for as unmetabolized toxin in urine. The fate of the remaining 84% (and of nearly the total dose when given orally) is unknown and remains the subject of further investigation.
Acknowledgements--The authors wish to thank H. J. B. Joubert and P. W. De Lange for technical assistance in the handling of laboratory animals and W. C. A. Gelderblom and M. E. Cawood for the provision of FB I . REFERENCES
Bezuidenhout S. C., Gelderblom W. C. A., Gorst-Allman C. P., Horak R. M., Marasas W. F. O., Spiteller G. and Vleggaar R. (1988) Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme. Journal of the Chemical Society, Chemical Communications 743-745. Cawood M. E., Gelderhlom W. C. A., Vleggaar R., Behrend Y., Thiel P. G. and Marasas W. F. O. (1991) Isolation of the fumonisin mycotoxins--a quantitative approach. Journal of Agricultural and Food Chemistry 39, 1958-1962. Gelderblom W. C. A., Jaskiewickz K., Marasas W. F. O., Thiel P. G., Horak R. M., Vleggaar R. and Kriek N. P. J. (1988) Fumonisins--novel mycotoxins with cancerpromoting activity produced by Fusarium moniliforme. Applied and Environmental Microbiology 54, 1806-1811. Gelderblom W. C. A., Kriek N. P. J., Marasas W. F. O. and Thiel P. G. (1991) Toxicity and carcinogenicity of the
279
Fusarium moniliforme metabolite, fumonisin Bi, in rats. Carcinogenesis 12, 1247-1251. Harrison L. R., Colvin B. M., Greene J. T., Newman L. E. and Cole J. R. (1990) Pulmonary edema and hydrothorax in swine produced by fumonisin Bt, a toxic metabolite of Fusarium moniliforme. Journal of Veterinary Diagnostic Investigation 2, 217-221. Kellerman T. S., Marasas W. F. O., Thiel P. G., Gelderblom W. C. A., Cawood M. and Coetzer J. A. W. (1990) Leukoencephalomalacia in two horses induced by oral dosing of fumonisin Bt . Onderstepoort Journal of Veterinary Research 57, 269-275. Marasas W. F. O., Kellerman T. S., Gelderblom W. C. A., Coetzer J. A. W., Thiel P. G. and Van der Lugt J. J. (1988) Leukoencephalomalacia in a horse induced by fumonisin B~, isolated from Fusarium moniliforme. Onderstepoort Journal of Veterinary Research 55, 197-203. Rheeder J. P., Marasas W. F. O., Thiel P. G., Sydenham E. W., Shephard G. S. and Van Schalkwyk D. J. (1992) Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei. Phytopathology 82, 353-357. Ross P. F., Rice L. G., Plattner R. D., Osweiler G. D., Wilson T. M., Owens D. L., Nelson H. A. and Richard J. L. (1991) Concentrations of fumonisin BI in feeds associated with animal health problems. Mycopathologia 114, 129-135. Shephard G. S., Sydenham E. W., Thiel P. G. and Gelderblom W. C. A. (1990) Quantitative determination of fumonisins Bt and B 2 by high-performance liquid chromatography with fluorescence detection. Journal of Liquid Chromatography 13, 2077-2087. Shephard G. S., Thiel P. G. and Sydenham E. W. (1992) Determination of fumonisin Bt in plasma and urine by high-performance liquid chromatography. Journal of Chromatography 574, 299-304. Sydenham E. W., Shephard G. S., Thiel P. G., Marasas W.F.O. and Stockenstr6m S. (1991) Fumonisin contamination of commercial corn-based human foodstuffs. Journal of Agricultural and Food Chemistry 39, 2014-2018. Sydenham E. W., Thiel P. G., Marasas W. F. O., Shephard G. S., Van Schalkwyk D. J. and Koch K. R. (1990) Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, southern Africa. Journal of Agricultural and Food Chemistry 38, 1900-1903. Thiel P. G., Marasas W. F. O., Sydenham E. W., Shephard G. S. and Gelderblom W. C. A. (1991a) The implications of naturally occurring levels of fumonisins in corn for human and animal health. Mycopathologia. In press. Thiel P. G., Marasas W. F. O., Sydenham E. W., Shephard G. S., Gelderblom W. C. A. and NieuwenhuisJ. J. (1991b) Survey of fumonisin production by Fusarium species. Applied Environmental Microbiology 57, 1089-1093. Thiel P. G., Shephard G. S., Sydenham E. W., Marasas W. F. O., Nelson P. E. and Wilson T. M. (1991c) Levels of fumonisins Bt and B2 in feeds associated with confirmed cases of equine leukoencephalomalacia. Journal of Agricultural and Food Chemistry 39, 109-111. Waynforth H. B. (1980) Experimental and Surgical Techniques in the Rat. p. 240. Academic Press, London.
