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A comparative study of monooxygenase activity in elasmobranchs and mammals: Activation of the model pro-carcinogen aflatoxin B1 by liver preparations of calf, nurse shark and clearnose skate
Camp. Biochem. PArsid. Printed in Great Bhin
Vol. 82C.
No. 2. pp. 255-257.
1985
0306.4492385 $3.00 + 0.00 I(‘ 1985 Pergamon Press Ltd
A COMPARATIVE STUDY OF MONOOXYGENASE ACTIVITY IN ELASMOBRANCHS AND MAMMALS: ACTIVATION OF THE MODEL PRO-CARCINOGEN AFLATOXIN B, BY LIVER PREPARATIONS OF CALF, NURSE SHARK AND CLEARNOSE SKATE A. B. BODINE,*
C. A. LUER~ and
S. GANGJEE*
*Department of Dairy Science. Clemson University, Clemson, SC 29631, USA. Telephone: (803) 656-3230 and SMote Marine Laboratory, Sarasota, FL. USA (Received 8 Mu): 1985) Liver microsome preparations of the elasmobranchs Ginglymostoma cirrutum (nurse shark) and Ruju eglunteriu (clearnose skate) were examined for monooxygenase activity using aflatoxin B, as substrate. 2. At equiprotein concentrations, elasmobranch microsomes were less than 20% as active as calf liver in producing mutagenic metabolites of aflatoxin B,. Abstract-l.
INTRODUCTION
Aflatoxin B, (AFB,) is the predominant homologue of a group of toxic bis-furanocoumarins which are produced as secondary metabolites by numerous toxigenic strains of Aspergillusjlavus and A. parasiticus (Stoloff, 1977). Aflatoxin B, has an LD,,, (p.0.) for most species of < 15 mg/kg body wt (Ciegler, 1975), and at the present time is considered to be the most potent naturally occurring carcinogen for the rat (Wogan, 1973). Numerous other species have been shown to be susceptible to aflatoxin-induced carcinogenesis including the trout, duckling, ferret, pig, tree shrew and guppy (Wogan, 1977) and in addition AFB, has been implicated as an etiologic factor in human cancer through the food chain in underdeveloped countries (Shank, 1971). In order to bind to cellular DNA and presumably initiate oncogenesis, AFB, must first be ‘*activated” to form an 8, 9 (or more recently renumbered 15, 16) epoxide which forms covalent adducts with DNA principally via the N7 position in guanines (Essigman, 1977). The requisite activation of AFB, occurs principally in the mixed function oxidase (MFO) conglomerate of the endoplasmic reticulum of the liver and possibly other organs (Stoloff, 1977). Thus, the ability to activate AFB, to the carcinogenic form coupled with auxilliary detoxification pathways for the epoxide (e.g. glutathione S-transferase, UDPglucuronyl transferase, epoxide hydrolase, etc.) play important roles in a species’ refractoriness to AFB,induced carcinogenesis (Degen and Neumann, 1981). Researchers have long been interested in the apparent resistance of elasmobranchs to neoplasia. Prior to the establishment of the Registry of Tumors in Lower Animals (Harshbarger, 1969) in 1965, only seven tumors in shark species were reported (Welling% ~__ Contribution No. 2391 from tural Experiment Station,
~the South Carolina AgriculClemson, SC, USA.
1969). Since the advent of the registry only nine tumors have been diagnosed from shark tissue, only two of which might be definitely identified as malignant. Other researchers in the study of elasmobranchs have reported few if any malignancies in thousands of sharks examined at necropsy (Sigel, 1977; Prieur et al., 1976; Walker et al., 1968). It would therefore appear that the elasmobranch is an ideal model for investigation of chemically-induced mutagenesis/ carcinogenesis. The purpose of this paper is to report a comparative study of MFO activation capacity in liver preparations of a mammalian and two elasmobranch species, using the model pro-carcinogen AFB,. MATERIALS AND METHODS
Aflatoxin B, was obtained from Sigma, St. Louis and checked for purity by TLC and reverse phase HPLC (Neal and Colley, 1978). All other reagents, e.g. NADP+, glucose 6-phosphate dehydrogenase, dimethyl sulfoxide, etc. were obtained from either Sigma, Aldrich, Milwaukee, or Tridom Fluka Hauppauge, NY and were the best quality available (reagent grade or better). Calves were obtained from the Clemson University dairy herd. All calves were male Holsteins approximately 3-5 months of age. Nurse sharks (Gingl;vmostomu cirrutum) were collected in the Florida Keys, while clearnose skates (Ruja eglanteriu) were obtained from nearshore Gulf of Mexico waters off Longboat Key, FL. Liver 9000 g postmitochondrial supematants were prepared by the methods outlined by Ames et al. (1975). and stored at -80°C until use (13 weeks). The histidine dependent mutant Salmonella typhimurium TA98 was obtained from Dr Bruce N. Ames, University of California at Berkeley, and was cultured, stored and checked for retention of genetic markers by methods outlined by Ames et al. (1975). Protein determinations were by the methods of Bradford (1976) using crystalline bovine serum albumin as standard. One milligram of microsomal protein (100,000 gpellet, 4’C. 1 hr) derived from the 9000 g postmitochondrial supernatant preparations of the calf, shark or skate was used as the source of mixed function oxidases. Early attempts to use 255
256
A. B.
BOD~NF
the 9000g supernatant as a source of eiasmobranch MFO resulted in extremely low spontaneous reversion rates and very sparse or completely absent background lawns. It was presumed that the low bacteria counts were the results of the very high lipid content of the elasmobranch 9000g supernatants thus necessitating the use of the microsomal pellet. The MFO enzyme source, phosphate buffer, NADPH regenerating system, 0.1 ml of-a i5hr culture of TA98 and either 0. 0.1. 0.2 or 0.4 UP or AFB, in 40 ul DMSO were incubated in small borosil;ate vials for 30 min at 37°C (for calves) or 30°C (for elasmobranchs) in a shaking water bath at 80 osc/min. Incubations were conducted in the dark to minimize light-induced destruction of AFB,. After incubation the vial contents were mixed with 2 ml of Ames top agar containing traces of histidine and biotin (Ames et al., 1975). vortexed briefly. and quickly poured onto the surface of Vogel-Bonner medium E plates and allowed to harden (about 20 mm). After hardening the plates were inverted and incubated at 37-C for 48 f 1 hr. After incubation the macroscopic revertant colonies were counted and the presence of spontaneous revertants and background lawn of non-revertants confirmed. RESULTS
The data for AFB,-induced reversion of TA98 as a result of activation by species liver preparations are depicted in Table I. The mean corrected (colony
count - spontaneous count) revertant counts vs concentration of toxin are also graphically illustrated in Fig. I. All corrected revertant counts are expressed on an equiprotein (1 mg microsomal protein) basis. The calf liver microsome preparations were approximately 5-fold more active in converting AFB, to the mutagenic form compared to microsome preparations of either elasmobranch. The calf liver data showed the typical linear dose response of revertant colonies as a function of carcinogen concentration and, in addition, even at the lowest concentration of AFB, tested (0.1 pg) there was more than a 4-fold increase in revertant colonies over control. The liver preparations of either elasmobranch showed little capacity to activate AFB, to the mutagenic form, nor was there evidence of a dose-response relationship. Indeed, even at the highest concentration of AFB, employed there was, only a 3-fold increase in the number of revertants over the control (spontaneous revertant coionies = 30 - 37). DISCUSSION
The very low monooxygenase activity observed in elasmobranch liver is in agreement with the reported Table
I. ARatoxin f~~~~~~~~~
Species __Calf (N = 8)
~~
Nurse shark (N=4) Clearnose (N=S)
skate
B, induced reverse mutation of Safmnneiln by liver preparation of several species AFB, (I@) 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4
No. h&&dine revertants* i_sHvlt 128 _+ 14 248 + 25 451 i49 30 * 5 30 ri: 4 66+_ 18 31+11 40* II x50* 17
*Corrected (tot31 No. - spontzmeous) tSEM-standard error of the mean.
No. revertants per nmoi AFB, tSEM 400 388 352 94 47 52 97 63 47 WI+ 10
et al
l
Calf
n Shark 0
Aflatoxin
Skate
6, +JI
Fig. 1. Dose response of revertants vs AFB, dose for calf, shark and skate liver preparations.
generally lower activities of MFO enzymes in elasmobranchs compared with mammals (Pohl et al., 1974). Many chemical carcinogens require metabolic activation in order to form a DNA-binding electrophile. The minimal monooxygenase activity observed for eIasmobranchs in these experiments would tend to spare cellular DNA from electrophilic attack. It has also been reported (Degen and Neumann 1978, 1981) that the glutathione S-transferases (EC 2.5.1.18) play a major role in phase II biotransformation of electrophilic species, e.g. AFB,-oxide by formation of glutathione adducts. Interestingly, additional work in this laboratory (unpublished data) has revealed that GST activity in nurse shark or clearnose skate is similar to the activity in calf liver (h 300-500 nmoles~min~mg with l~hloro-2,4-dinitrobenzene as substrate). The relatively high phase II activity in conjunction with low pro-carcinogen activation capability of elasmobranch liver preparations would appear to play a role in resistance of elasmobranchs to chemical carcinogenesis. REFERENCES
Ames B. N., McCann J. and Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with the Sulmonellu/mammalian microsome mutagenicity test. Mutation Res. 31, 347-361. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anaf.vt. Biochem. 72, 248-251. Ciegler A. (I 975) Mycotoxins: occurrence, chemistry, bioloeical activitv. Llovdia 38, 21-35. De&n G. and Newman” H. G. (1978) The major metabolite of allatoxin B, in the rat is a glutathione conjugate. Chem. biol. Interactions 22, 239.-255. Degen G. and Neumann I-I. G. (1981) Differences in aflatoxin B, susceptibility of rat and mouse are correlated with the capability in citro to inactivate aflatoxin B, epoxide. Carcinogenesis 2, 299-304. Essigman J. M.. Croy R. G.. Nagzan W. F., Busby V. N., Reinhold V. N., Buch G. and Wogan G. N. (1977) Structural identification of the principle aflatoxin B,DNA adduct formed by aflatoxin B, in t:itro. Proc. mtn. Acad. Sci. U.S.A. 74, 1870-1874. Harshberger J. C. (1969) The registry of tumors in lower animals. Monograph natn. Cancer Inst. 31, 11.
Monooxygenase Neal G. E. and Colley P. J. (1978) Some high performance liquid chromotographic studies of the metabolism of aflatoxin B, by rat liver microsomal preparations. Biochem. J. 17, 839-844. Prieur D. J., Fenstermacher J. D. and Guarino A. M. (1976) A choroid plexus papilloma in an elasmobranch Squalus acanthias. J. natn. Cancer Inst. 56, 1207-1209. Shank R. C. (1971) Dietary aflatoxin loads are the incidence of human hepatocellular carcinoma in Thailand. In Symposium on Mycotoxins in Human Health (Edited by Purchase I. F. H.), pp. 245-262. MacMillan, London. Sigel M. (1977) Neoplasms in aquatic animals in aquatic pollutants and biological effects with emphasis on neoplasia. Ann. N. Y. Acad. Sci. 298-319.
in elasmobranchs
257
Stoloff L. (1977) Aflatoxins-an overview. In Mycotoxins in Human and Animal Health, pp. l-28. Pathotox, Park Forest South. Walker M., Zubrod G. and Gilbert P. W. (1968) Activities report: registry of tumors in lower animals. Accession No. 212. Wellings S. R. (1969) Neoplasms and related disorders of invertebrate and lower vertebrate animals. Monograph naln. Cancer Inst. pp. 55-121. Wogan G. N. (1973) Aflatoxin carcinogenesis. In Methods in Cancer Research, Vol. VII. pp. 3099344. Academic Press, New York. Wogan G. N. (1977) Mode of action of aflatoxins. In Mycotoxins in Human and Animal Health, pp. 29-36. Pathotox, Park Forest South.
Vol. 82C.
No. 2. pp. 255-257.
1985
0306.4492385 $3.00 + 0.00 I(‘ 1985 Pergamon Press Ltd
A COMPARATIVE STUDY OF MONOOXYGENASE ACTIVITY IN ELASMOBRANCHS AND MAMMALS: ACTIVATION OF THE MODEL PRO-CARCINOGEN AFLATOXIN B, BY LIVER PREPARATIONS OF CALF, NURSE SHARK AND CLEARNOSE SKATE A. B. BODINE,*
C. A. LUER~ and
S. GANGJEE*
*Department of Dairy Science. Clemson University, Clemson, SC 29631, USA. Telephone: (803) 656-3230 and SMote Marine Laboratory, Sarasota, FL. USA (Received 8 Mu): 1985) Liver microsome preparations of the elasmobranchs Ginglymostoma cirrutum (nurse shark) and Ruju eglunteriu (clearnose skate) were examined for monooxygenase activity using aflatoxin B, as substrate. 2. At equiprotein concentrations, elasmobranch microsomes were less than 20% as active as calf liver in producing mutagenic metabolites of aflatoxin B,. Abstract-l.
INTRODUCTION
Aflatoxin B, (AFB,) is the predominant homologue of a group of toxic bis-furanocoumarins which are produced as secondary metabolites by numerous toxigenic strains of Aspergillusjlavus and A. parasiticus (Stoloff, 1977). Aflatoxin B, has an LD,,, (p.0.) for most species of < 15 mg/kg body wt (Ciegler, 1975), and at the present time is considered to be the most potent naturally occurring carcinogen for the rat (Wogan, 1973). Numerous other species have been shown to be susceptible to aflatoxin-induced carcinogenesis including the trout, duckling, ferret, pig, tree shrew and guppy (Wogan, 1977) and in addition AFB, has been implicated as an etiologic factor in human cancer through the food chain in underdeveloped countries (Shank, 1971). In order to bind to cellular DNA and presumably initiate oncogenesis, AFB, must first be ‘*activated” to form an 8, 9 (or more recently renumbered 15, 16) epoxide which forms covalent adducts with DNA principally via the N7 position in guanines (Essigman, 1977). The requisite activation of AFB, occurs principally in the mixed function oxidase (MFO) conglomerate of the endoplasmic reticulum of the liver and possibly other organs (Stoloff, 1977). Thus, the ability to activate AFB, to the carcinogenic form coupled with auxilliary detoxification pathways for the epoxide (e.g. glutathione S-transferase, UDPglucuronyl transferase, epoxide hydrolase, etc.) play important roles in a species’ refractoriness to AFB,induced carcinogenesis (Degen and Neumann, 1981). Researchers have long been interested in the apparent resistance of elasmobranchs to neoplasia. Prior to the establishment of the Registry of Tumors in Lower Animals (Harshbarger, 1969) in 1965, only seven tumors in shark species were reported (Welling% ~__ Contribution No. 2391 from tural Experiment Station,
~the South Carolina AgriculClemson, SC, USA.
1969). Since the advent of the registry only nine tumors have been diagnosed from shark tissue, only two of which might be definitely identified as malignant. Other researchers in the study of elasmobranchs have reported few if any malignancies in thousands of sharks examined at necropsy (Sigel, 1977; Prieur et al., 1976; Walker et al., 1968). It would therefore appear that the elasmobranch is an ideal model for investigation of chemically-induced mutagenesis/ carcinogenesis. The purpose of this paper is to report a comparative study of MFO activation capacity in liver preparations of a mammalian and two elasmobranch species, using the model pro-carcinogen AFB,. MATERIALS AND METHODS
Aflatoxin B, was obtained from Sigma, St. Louis and checked for purity by TLC and reverse phase HPLC (Neal and Colley, 1978). All other reagents, e.g. NADP+, glucose 6-phosphate dehydrogenase, dimethyl sulfoxide, etc. were obtained from either Sigma, Aldrich, Milwaukee, or Tridom Fluka Hauppauge, NY and were the best quality available (reagent grade or better). Calves were obtained from the Clemson University dairy herd. All calves were male Holsteins approximately 3-5 months of age. Nurse sharks (Gingl;vmostomu cirrutum) were collected in the Florida Keys, while clearnose skates (Ruja eglanteriu) were obtained from nearshore Gulf of Mexico waters off Longboat Key, FL. Liver 9000 g postmitochondrial supematants were prepared by the methods outlined by Ames et al. (1975). and stored at -80°C until use (13 weeks). The histidine dependent mutant Salmonella typhimurium TA98 was obtained from Dr Bruce N. Ames, University of California at Berkeley, and was cultured, stored and checked for retention of genetic markers by methods outlined by Ames et al. (1975). Protein determinations were by the methods of Bradford (1976) using crystalline bovine serum albumin as standard. One milligram of microsomal protein (100,000 gpellet, 4’C. 1 hr) derived from the 9000 g postmitochondrial supernatant preparations of the calf, shark or skate was used as the source of mixed function oxidases. Early attempts to use 255
256
A. B.
BOD~NF
the 9000g supernatant as a source of eiasmobranch MFO resulted in extremely low spontaneous reversion rates and very sparse or completely absent background lawns. It was presumed that the low bacteria counts were the results of the very high lipid content of the elasmobranch 9000g supernatants thus necessitating the use of the microsomal pellet. The MFO enzyme source, phosphate buffer, NADPH regenerating system, 0.1 ml of-a i5hr culture of TA98 and either 0. 0.1. 0.2 or 0.4 UP or AFB, in 40 ul DMSO were incubated in small borosil;ate vials for 30 min at 37°C (for calves) or 30°C (for elasmobranchs) in a shaking water bath at 80 osc/min. Incubations were conducted in the dark to minimize light-induced destruction of AFB,. After incubation the vial contents were mixed with 2 ml of Ames top agar containing traces of histidine and biotin (Ames et al., 1975). vortexed briefly. and quickly poured onto the surface of Vogel-Bonner medium E plates and allowed to harden (about 20 mm). After hardening the plates were inverted and incubated at 37-C for 48 f 1 hr. After incubation the macroscopic revertant colonies were counted and the presence of spontaneous revertants and background lawn of non-revertants confirmed. RESULTS
The data for AFB,-induced reversion of TA98 as a result of activation by species liver preparations are depicted in Table I. The mean corrected (colony
count - spontaneous count) revertant counts vs concentration of toxin are also graphically illustrated in Fig. I. All corrected revertant counts are expressed on an equiprotein (1 mg microsomal protein) basis. The calf liver microsome preparations were approximately 5-fold more active in converting AFB, to the mutagenic form compared to microsome preparations of either elasmobranch. The calf liver data showed the typical linear dose response of revertant colonies as a function of carcinogen concentration and, in addition, even at the lowest concentration of AFB, tested (0.1 pg) there was more than a 4-fold increase in revertant colonies over control. The liver preparations of either elasmobranch showed little capacity to activate AFB, to the mutagenic form, nor was there evidence of a dose-response relationship. Indeed, even at the highest concentration of AFB, employed there was, only a 3-fold increase in the number of revertants over the control (spontaneous revertant coionies = 30 - 37). DISCUSSION
The very low monooxygenase activity observed in elasmobranch liver is in agreement with the reported Table
I. ARatoxin f~~~~~~~~~
Species __Calf (N = 8)
~~
Nurse shark (N=4) Clearnose (N=S)
skate
B, induced reverse mutation of Safmnneiln by liver preparation of several species AFB, (I@) 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4
No. h&&dine revertants* i_sHvlt 128 _+ 14 248 + 25 451 i49 30 * 5 30 ri: 4 66+_ 18 31+11 40* II x50* 17
*Corrected (tot31 No. - spontzmeous) tSEM-standard error of the mean.
No. revertants per nmoi AFB, tSEM 400 388 352 94 47 52 97 63 47 WI+ 10
et al
l
Calf
n Shark 0
Aflatoxin
Skate
6, +JI
Fig. 1. Dose response of revertants vs AFB, dose for calf, shark and skate liver preparations.
generally lower activities of MFO enzymes in elasmobranchs compared with mammals (Pohl et al., 1974). Many chemical carcinogens require metabolic activation in order to form a DNA-binding electrophile. The minimal monooxygenase activity observed for eIasmobranchs in these experiments would tend to spare cellular DNA from electrophilic attack. It has also been reported (Degen and Neumann 1978, 1981) that the glutathione S-transferases (EC 2.5.1.18) play a major role in phase II biotransformation of electrophilic species, e.g. AFB,-oxide by formation of glutathione adducts. Interestingly, additional work in this laboratory (unpublished data) has revealed that GST activity in nurse shark or clearnose skate is similar to the activity in calf liver (h 300-500 nmoles~min~mg with l~hloro-2,4-dinitrobenzene as substrate). The relatively high phase II activity in conjunction with low pro-carcinogen activation capability of elasmobranch liver preparations would appear to play a role in resistance of elasmobranchs to chemical carcinogenesis. REFERENCES
Ames B. N., McCann J. and Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with the Sulmonellu/mammalian microsome mutagenicity test. Mutation Res. 31, 347-361. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anaf.vt. Biochem. 72, 248-251. Ciegler A. (I 975) Mycotoxins: occurrence, chemistry, bioloeical activitv. Llovdia 38, 21-35. De&n G. and Newman” H. G. (1978) The major metabolite of allatoxin B, in the rat is a glutathione conjugate. Chem. biol. Interactions 22, 239.-255. Degen G. and Neumann I-I. G. (1981) Differences in aflatoxin B, susceptibility of rat and mouse are correlated with the capability in citro to inactivate aflatoxin B, epoxide. Carcinogenesis 2, 299-304. Essigman J. M.. Croy R. G.. Nagzan W. F., Busby V. N., Reinhold V. N., Buch G. and Wogan G. N. (1977) Structural identification of the principle aflatoxin B,DNA adduct formed by aflatoxin B, in t:itro. Proc. mtn. Acad. Sci. U.S.A. 74, 1870-1874. Harshberger J. C. (1969) The registry of tumors in lower animals. Monograph natn. Cancer Inst. 31, 11.
Monooxygenase Neal G. E. and Colley P. J. (1978) Some high performance liquid chromotographic studies of the metabolism of aflatoxin B, by rat liver microsomal preparations. Biochem. J. 17, 839-844. Prieur D. J., Fenstermacher J. D. and Guarino A. M. (1976) A choroid plexus papilloma in an elasmobranch Squalus acanthias. J. natn. Cancer Inst. 56, 1207-1209. Shank R. C. (1971) Dietary aflatoxin loads are the incidence of human hepatocellular carcinoma in Thailand. In Symposium on Mycotoxins in Human Health (Edited by Purchase I. F. H.), pp. 245-262. MacMillan, London. Sigel M. (1977) Neoplasms in aquatic animals in aquatic pollutants and biological effects with emphasis on neoplasia. Ann. N. Y. Acad. Sci. 298-319.
in elasmobranchs
257
Stoloff L. (1977) Aflatoxins-an overview. In Mycotoxins in Human and Animal Health, pp. l-28. Pathotox, Park Forest South. Walker M., Zubrod G. and Gilbert P. W. (1968) Activities report: registry of tumors in lower animals. Accession No. 212. Wellings S. R. (1969) Neoplasms and related disorders of invertebrate and lower vertebrate animals. Monograph naln. Cancer Inst. pp. 55-121. Wogan G. N. (1973) Aflatoxin carcinogenesis. In Methods in Cancer Research, Vol. VII. pp. 3099344. Academic Press, New York. Wogan G. N. (1977) Mode of action of aflatoxins. In Mycotoxins in Human and Animal Health, pp. 29-36. Pathotox, Park Forest South.