AN ABSTRACT OF THE THESIS OF GRANT LOUIS SCHOENHARD (Name) in FOOD SCIENCE (Major) Title: AFLATOXIN B for the DOCTOR OF PHILOSOPHY (Degree) presented on March 15, 1974 (Date) METABOLISM BY RAINBOW TROUT (SALMO GAIRDNERI) ■-*' '' ■ J'SV Abstract approved: ^ ' J-' Jf 5^5^ „ . „ . Donald jyLee T ■&* ' ■~~ T^ " r*"r. Aflatoxins B,, Q, and aflatoxicol, R F, but not B- were acti-< 1 l o L vated in the presence of NADPH by the 105, 000 x g pellet from rainbow trout (Salmo gairdner-i), Mt, Shasta strain, liver to products lethal to Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1). Electronmicros- copy confirmed the microsomal fraction primarily contained smooth endoplasmic reticulum. An NADPH mixed function oxidase with neo- tetrazolium reductase and aldrin epoxidase activity was present in the microsonaes. The activity of the oxidase was reduced parallel to a decrease in lethal factor production by piperor^yl butoxide, NADPH deprivation, heat treatment and certain diets. microsomes with 14 After incubation of C labeled B , the activity was found in unaltered B and three extremely polar metabolites designated NM (3. 6-5. 3%), B A (1-5%) and M^A (< 1%). They were eliminated or reduced along with microbial lethality when cytosine and cysteine were added to the incubation media. The structural requirement for the vinyl ether of B,, R F and 1 o Q and the nature of the enzymatic reaction were consistent with the hypothesis that the conapounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B^. subtilis. Aflatoxicol, R F, was isolated from liver homogenates and was o apparently fornaed by an NADPH-dependent soluble enzyme of the 105, 000 x g supernatant from rainbow trout. R F and its diastereoo mer, R R, were prepared by chemical reduction of B . identity was confirmed by UV and mass spe chrome try. Their The ten-day LD50 value was 0. 66 mg/kg for R F compared to 0. 46 mg/kg for B.. No mortality was observed, from R R at equivalent doses. o Similar gross and microscopic pathological changes were observed for B , R F and R R. The degree of damage paralleled the LD50 values. After eight months of feeding, 20 ppb of B and R F and 36. 6 ppb R R gave 56. 3%, 26. 2% and 0%. incidence of hepatoma. Addition of 50 ppm of cyclopropenoid fatty acids increased the incidence to 96. 3%, 93. 8% and 55% for Bn1, R o F and R o R, - ■•'/".'., It was concluded that aflatoxicol was not an effective means of detoxication of B , but instead extended the presence of a potentially toxic and carcinogenic compound. Aflatoxin B Metabolism by Rainbow Trout (Salmo gairdneri) by Grant Louis Schoenhard A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1974 APPROVED: Associate Professor of Foo^Sciencpgand Technology in charge of major Head of Departnnent of Food Science and Technology ,1- f - ,»-■■-, , „■,- w i ^r, i^<l9,y ly Dean of Grarauate School Date thesis is presented Typed by Mary Jo Stratton for March 15, 1974 Grant Louis Schoenhard ACKNOWLEDGMENT The author wishes to express his gratitude to Dr. Donald J. Lee for guidance and assistance during this investigation. The author expresses appreciation to Professor Russell O. Sinnhuber for his interest and support. The author i$ grateful to Dr. P. E. Bishop for assistance in establishing the microbial assay, Dr. J, S. Lee and Dr. D. J. Reed for use of their laboratories, Dr. N. E. Pawlowski for the rpethod and assistance with the preparation of the crude diastereomers of aflatoxicol, Dr. L. C. Terriere for the analysis of dieldrin, and Professor J. H. Wales for guidance in interpretation and photomicrographs of histological preparations. The author thanks Mr. S. E. Howell, Mr. P, Kwan, Mrs. L. J. Hunter, Dr. L. M. Libbey, Mr. T. Will, Mr, R. A. Foster and Miss L. K. Moreland foi; technical assistance on various aspects of the study. Most of all, the author would like to thank his wife, Alice, for her complete support, technical aid with the acute toxicity trial and assistance in the preparation of the manuscript. This research was supported by Public Health Service Training Grant FD00010 and by Public Health Service Research Grants ES00541 and ES00256. The author expresses his gratitude to this agency for making the study possible. TABLE OF CONTENTS .Page INTRODUCTION 1 LITERATURE REVIEW 2 Aflatoxin Toxicity and Carcinogenicity of Aflatoxin Bj Biochemical Effects of Aflatoxin Bj DNA Binding and Metabolism Mitochondrial Respiration Protein RNA Aflatoxin Bj Metabolism Species Comparison Activation Xenobiotic Metabolism by Fish Chemical Initiation of Cancer Carcinogens as Mutagens EXPERIMENTAL Metabolism Studies Centrifugation Fractions of Liver Homogenates Preparation of Incubation Mixtures Calculation of Mierobial Viability Isolation and Identification of Aflatoxicol Aflatoxicol from Trout Preparations Aflatoxicol Diastereomers from Chemical Reduction of Bi Extraction, Purification and Identification of MjA, B2aA and NM Acute Toxicity Trial Chronic Toxicity and Carcinogenicity Trial RESULTS AND DISCUSSION Mierobial Assay Enzymatic Conditions for Lethal Factor Production Metabolism of Aflatoxin Effect of Inhibitors Extraction of Metabolites Lethality and Identification of Metabolites 2 2 6 6 6 7 9 10 10 14 16 20 22 25 25 25 28 30 30 30 31 32 34 36 37 37 39 50 50 52 56 -Page Acute Toxicity Trial Mortality and General Observations Histological Observations Liver Kidney Chronic Toxicity and Carcinogenicity Trial Liver Chronic Toxicity Hepatoma Kidney Chronic Toxicity Significance of Metabolism of B. 1 Areas for Future Study 65 65 68 68 68 74 74 74 77 77 77 77 86 SUMMARY AND CONCLUSIONS 88 BIBLIOGRAPHY 90 APPENDICES Appendix I, Appendix H. Appendix III. Appendix IV. Appendix V. Appendix VI. Appendix VII. Appendix VIII. Appendix Appendix Appendix Appendix IX. X. XI, XII. Microbial Assay Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1); Partial Confirmation of Phenotype Krebs Ringer's Solution Adjusted for Fish NADPH Generating Systems Quantitation of Aflatoxins Bi, B2, RQF, ROR and Qj Thin Layer Chromatography: Preparative and Analytical Purification of Aflatoxicol Diastereomers RrtF and R R by Column o o Chromatography Mass Spectral Analysis of Aflatoxicol Diastereomers R.FandR R Neotetrazolium Reductase Assay Aldrin Epoxidation Assay Diets Liquid Scintillation Counting Fluor Solutions 120 122 123 124 125 127 129 131 132 133 134 135 LIST OF TABLES Table Page 1 Species Comparison of B 2 Cornparative Microsomal Activity. 19 3 Carcinogens as Mutagens. 23 4 Lethal Factor Production. 38 5 Effect of Diet a^nd Feeding on Lethal Factor Production by 20, 000 x g Supernatant. 42 Effect of Incubation Temperature and Prior Heat Treatment. 44 Effect of 20, 000 x g Supernatant of Acetone Powder Preparation. 44 Lethal Factor Production and NT Reductase Activity of 20, 000 x g Supernatant and Ca++ Precipitated Microsomes. 46 Lethal Factor Production and. NT Reductase Activity of 105, 000 x g Fractions. 47 Effect of Piperonyl Butoxide on Aldrin Epoxidation and Lethal Factor Production. 49 Effect of Selected Nucleophiles on Lethal Factor Production. 53 12 Metabolites by TLC. 55 13 Microbial Lethality of Unactivated and Activated Aflatpxicol Diastereomers. 60 Comparative Microbial Lethality of B , Q and B,, 60 6 7 8 9 10 11 14 15 Metabolites. Mortality in Rainbow Trout 10 Days after Intraperitoneal Administration of Aflatoxins B,, R F and R R. 1 o o 11 66 Table 16 Page Incidence of Hepatoma at Four and Eight Months from Start of Feeding Trial. 78 LIST OF FIGURES Figure Page 1 Aflatoxin structures. 2 Flow diagram of liver fractionation by centrifugation. 26 3 Ca 27 4 Incubation mixtures. 29 5 Calculation of miqrobial viability. 30 6 Loss of viability as a function of microbial incubation time. 40 Loss of viability as a function of B concentration. 41 Top layer 105, 000 x g pellet composed primarily of smooth endoplasmic reticulum. 51 Hypothetical Schiff base formation of amino acids with one of the resonance forms of the phenolate ion. 62 11 Liver from trout dosed with 0. 4 mg/kg B . 69 12 Liver from trout dosed with 0. 56 mg/kg R F. 69 13 Liver from trout dosed with 1. 27 mg/kg R R. 70 14 Normal trout liver. 71 15 Normal trout kidney. 71 16 Kidney from trout dosed with 0. 4 mg/kg B . 72 17 Kidney from trout dosed with 1. 27 mg /kg R R. 72 18 Kidney from trout dosed with 0. 85 mg/kg R F. 73 19 Kidney from trout dosed with 0. 56 mg/kg R R. 73 7 8-9 10 precipitation of liver microsornes. 3 Figure 20 21 22 23 24 25 26-27 28 29 30 31 32 33 34 Page Liver from trout fed 20 ppb B four months. for 75 Liver from trout fed 20 ppb R F for four months. 75 Liver from trout fed 36. 6 ppb R R for four months. 76 Liver from trout fed 50 ppm CPFA for four months. 76 Liver from trout fed 20 ppb B CPFA for four months. 79 and 50 ppm Liver from trout fed 36. 6 ppb R R and 50 ppm CPFA for four months. 0 79 Liver from trout fed 20 ppb R F ^rad 50 ppm CPFA for four months? 80 Kidney from trout fed 20 ppb R F for four months. 81 Kidney from trout fed 36. 6 ppb R R for four months. 81 Kidney from trout fed 50 ppm CPFA for four months. 82 Kidney from trout fed 20 ppb B. and 50 ppm CPFA for four months. 82 Kidney frona trout fed 20 ppb R F and 50 ppm CPFA for four months. 83 Kidney from trout fed 36. 6 ppb R R and 0 50 ppm CPFA for four months, 83 Hypothetical pathways of B metabolism. 84 and R F AFLATOXIN Bl METABOLISM BY RAINBOW TROUT (SALMO GAIRDNERI) INTRODUCTION Aflatoxin B.. is extremely toxic and among the most potent carcinogens. It has been found in many foods consumed by man and domestic animals. Various treatments of food have been tried to reduce the toxic and carcinogenic hazard. These efforts could be aided by knowledge of the structural requirements foj: the ultimate biological activity of aflatoxin B.. As indicated in the literature review, B. has not been shown to inhibit DNA template activity, DNA dependent RNA polymerase or malignantly transform cell cultures in vitro without the presence of an active microsomal NADPH-dependent mixed function oxidase. Hypophysectomy and various diets protect rats against B hepatoma. B induced is activated to a product lethal and mutagenic to Salmonella typhimurium in the presence of an active microsomal preparation. Various workers have hypothesized that B is meta- bolically activated to a highly reactive and unstable rnetabolite as the ultimate toxin and/or carcinogen. The objectives of this study were to determine whether rainbow trout (Salmo gairdneri), Mt. Shasta strain, activated B and to evalu- ate the structure-'activity relationship of the metabolites for toxicity and carcinogenicity. LITERATURE REVIEW Aflatoxin Well over 1, 000 publications have appeared on aflatoxin in the last decade. Several comprehensive reviews including the areas not reviewed below, such as etiology, biosynthesis and chemistry, are available (57, 104, 121, 260). Toxicity and Carcinogenicity of Aflatoxin B 1 Many of the early investigations with aflatoxin B (Figure 1) were concerned with its toxicity and carcinogenicity in a wide variety of species (57, 121), A hyman death.occurred after ingestion of aflatoxin B1 contaminated food. Toxin was isolated and identified by spectral analysis from the severely necrotic liver (33). The acute LDSOs for two species of macaque monkey were 2.2 mg/kg (Macaca irus) and 7. 8 rng/Kg (Macaca fascicularis) accompanied by massive liver damage (240, 265). Toxic hepatitis similar to that found in man in regions of the world with B contamination of food was found in African monkeys (Cereopithecus aethiops) administered 0. 01-0. 1 mg daily until death (8), Initial attempts to induce carcinoma in nonhuman primates were unsuccessful (73, 205). after prolonged B Recently primary liver carcinoma was induced administration (3, 123). _A OH iX ^ o '^ CK^^ OCH -. •OCH3 Aflatoxin Mj Aflatoxin B j o A o \n ua ^o-^-o-^s^OH Aflatoxin Pj Aflatoxin Qj o o OH J o Sj^o-^-^OCH- ^OCH3 Aflatoxin B2 Aflatoxicol (R0) ^o' ^o- ^-^ "OCH3 Tetrahydrodeoxoaflatoxin Bj (THDBj) o o r-r—fV" HO'N5Ao'^<^oCH3 Aflatoxin B'Za HO HO o Aflatoxin B, -2, 3-oxide o X0X0XJ< OCH, 2, S-dihydro-Z, 3-dihydroxy-aflatoxin B. Figure 1. Aflatoxin structures. 4 Typical LDSOs for mature rats were 7 mg/kg (47) and 5 mg/kg (302). Toxicity was markedly reduced or absent in prepubertal, castrated (245) or hypophysectomized (122) male rats and in nonpregnant females (50), The same treatments resulted in a reduction in hepatoma. A dietary level of 15 ppb produced a 100% incidence of hepatoma in rats (302). Renal neoplasms also occurred (156, 188). B1 or a metabolite reaching the embryo of rats through the placenta produced inflammatory, hyperplastic and neoplastic liver changes (124). Mature mice were highly resistant to toxicity (88, 224, 288). Hepatoma (202, 288) and subcutaneous carcinoma (86) have been induced. Newborn nnice were rnore prone, in contrast to females, to lethal effects and hepatoma (288). Early reports (79) suggested sheep, like mice, were resistant to the toxic effects. More recently an reported for wethers (15). 1JD50 of 20 mg/kg was Pretreatment with DDT and B the sensitivity of rams to LD50 doees (14). decreased Neoplasms have been found in three sheep over a five-year trial (164). For rainbow trout, a crude preparation of B gave an LD50 of 0. 5-1. 0-mg/kg- (131) while an LD50 of 0. 8 mg/kg was obtained for pure B (28). Over a 12-month period 4. 0 ppb B toma incidence (269). gave a 15% hepa- Tumor growth was greatly enhanced by sterculic acid (157, 158, 269, 270). Wunder (304) observed that sexually m,ature male rainbow trout were resistant to hepatoma compared to mature females and immature fish. Many microorganisms were sensitive to B (42, 46, 79, 160). Their response was genetic, i. e. , DNA inhibition, phage induction, or nongenetic expressed as growth inhibition. The minimal effective concentration was approximately 1 |j.g/ml for genetic responses and in excess of 30 (xg/ml for growth inhibition. Genetic effects including mitotic inhibition, DNA inhibition and chromosome aberrations and nongenetic effects including growth inhibition, cell degeneration and cell destruction were observed at less than litg/nrtloCBj. for a variety of cell cultures (79, 89, 98, 160, 162, 179, 206, 207, 208). Cell cultures of rat liver have been malignantly transformed in vitro and successfully back-trans planted in newborn rats producing carcinoma (284). Many of the species discussed with respect to their sensitivity to B have been considered for bioassays of the toxin (80, .160). The chick embryo assay (56, 133, 225, 286, 287) has been widely used because it produces characteristic lesions, has a reliable doseresponse curve, and is sensitive, fast and simple. The structure-activity relationship of aflatoxin for carcinoma has been studied with analogs in trout (17), rats (299) and cell cultures (99). In each case carcinoma required the unsaturation of the dihydrofurofuran moiety of B and was dependent on the degree of unsaturation and substituents on the lactone moiety. Biochemical Effects of Aflatoxin Bj DNA Binding and Metabolism. Wogan (298) hypothesized that direct interaction of B.. with DNA was the initial and critical event for expression of toxicity and carcinogenicity. B1 was bound in vitro to native double-strand helical calf-thymus DNA and to a lesser extent to heat-denatured single- strand calf-thymus DNA, but not to enzy- matically hydrolyzed calf-thymus DNA (31, 58, 59, 60, 61, 273). in vitro DNA-B The complex was easily broken by Sephadex G-50 chroma- tography (60) suggesting a weak non-covalent interaction. Fluorescence polarization studies confirmed that the interaction was weak (234). charge-transfer interaction between B A and DNA was postulated (203). It was suggested that DNA-strand breakage of HeLa cells was a direct action of the B with 3 HB present (285). (167) and with 14 CB In vivo binding to rat DNA was found to trout nucleic acids (16). Inhibition of DNA synthesis was reported for regenerating rat liver after partial hepatectomy (106, 119) and for non-regenerating rat liver (74, 75, 249). Mitochondrial Respiration. the major site of B toxicity. Mitochondria have been proposed as The B1 mediated decrease in mito- chondrial dehydrogenases and electron transfer catalysts in ducklings 7 and chickens was suggested as the toxic event in mitochondria (39, 40). B administered to rats produced swelling of mitochondria from liver and kidney, but not in those from heart or testes (18). Oxygen con- sumption and phosphorylation were depressed in mitochondrial preparations from rats administered 0. 45 mg B /kg body weight (278). In contrast, other workers (58) reported no change in respiratory capacity and P/O ratios with mitochondria from rats dosed with 7 mg B •■ /kg body weight. Aflatoxin B ■, inhibited succinate dehydrogenase of rat liver mitochondria (276). Whole homogenates and mitochondria from livers of several animal s pecies were examined for the hi vitro and in vivo effects of B, (135). It inhibited oxygen uptake in vivo but not in vitro in each species studied except weanling rats. in vitro effect on P/O ratios from male mice. tions of 2. 5-4. 8 x 10" There was no Aflatoxin B, concentra- M resulted in a 25-44% inhibition of electron transport inactively respiring rat liver mitochondria (92, 93). A maximum of 63% inhibition occurred with submitochondrial particles (SMP ) obtained by drastic sonication of liver mitochondria. The major site of inhibition was between cytochromes b and c^ (or c). Mitochondria and SMP s of protein deprived animals (5% casein semipurified diet) had similar respiratory control ratios to normal animals (20% casein diet). The protein deprived animals had a 30-50% reduc- tion in B. mediated inhibition. Protein. After B, was mix$d with calf-thymus histones in vitro, a shift in the B, UV spectrum (273) and an increase in viscosity of the mixture (31) was observed suggesting B-y was bound to the histones. Injection of rainbow trout with 400 jig B /kg body weight resulted in a 65% loss of histone and acidic proteins relative to DNA within 12 hours (55). Administration of B i to rats produced within 15 minutes an increase in the rate of deacetylation of histone fractions F2A1 and F3 (96). In vitro inhibition of amino acid incorporation by B, was observed in rat liver slices (58, 59, 272), duckling liver slices (272) and HeLa cell cultures (134). In vivo incorporation of C leucine into total liver protein was inhibited by administration of B-p 50% in monkeys (Macaca irus) (239), but no inhibition was seen in rats (58, 263, 297). Several semi-purified protein preparations isolated from rats after Bi treatment were inhibited: (1) tryptophan pyrrolase (58, 59, 110, 301), (2) zoxazolamine hydroxylase (226), and (3) prothrombin (24). The inhibition of protein synthesis was due in cell cultures to a direct action oh the polysomes (116). The stimulation of microsomal hydrox- ylase synthesis ar^d activation was similar to benzo[a] pyrene but of smaller magnitude due to protein inhibition (129). Nuclear DNAase II, assayed in vitro, was activated in the rat but not in the mouse (220, 221, 223, 257) after in vivo administration of B, and no interaction was seen by direct addition of B 1 to the enzyme assay (220, 221). The specific activity of liver lysosomal acid DNAase was markedly decreased by B.. in vitro (222). Ribonuclease activity from treated rats was not affected (97). Interaction of B. with acetate thiokinase was suggested to explain the large reduction of 1-^C-acetate incorporation into skin total lipids (172). In chickens, uridine diphosphate glucose-rglycogen 9 transglucosylase, glycogen synthetase and glycogen phosphorylase activity was reduced, but the hexose monophosphate shunt dehydrogenase activities were elevated (266). A shift in tissue-specific lactic dehydrogenases was found in the rat (5, 159) while no change was observed in rainbow trout (281). The early appearance of a-fetoprotein with B induced hepatoma (149, 150) has been used to assess the incidence of hepatoma among human populations exposed to dietary B RNA. Administration of B (238). to rats resulted in a dramatic decrease in RNA synthesis in regenerating liver (151, 152) and in intact liver (5 9, 194, 273). B Inhibition occurred within 15 minutes of administration and remained up to five days (110, 111). effect was seen with B The same added to incubating rat liver slices (59, 61). It was hypothesized that the decrease in RNA synthesis resulted from inhibition of DNA dependent RNA polymerase. found in nuclei isolated from B dosed rats (61, 111, 118). inhibition was time and dose dependent (227). 255). Lack of inhibition when B enantiomer of B The polymerase was not inhibited (201). inhibited with mouse liver slices incubated with B . tion of RNA polymerase with B The Precursor incorporation into 28S and 18S nuclear RNA was suppressed (112). isolated from mice dosed with B Inhibition was But, it was In vitro incuba- was not inhibitory (146, 198, 250, was replaced with the synthetic (229), B_ or tetrahydrodeoxoaflatoxin B1 (282) 10 demonstrated an obligate structural requirement. cluded that in vivo inhibition with B Wogan (2 96) con- was possible because metabblic transformation was feasible while neither was true in vitro. It was suspected, because of the marked effect on nuclear RNA synthesis, that cytoplasmic RNA would also be altered. Polysome disaggregation was demonstrated (228, 250, 252, 294). This was reported to occur because B blocked the steriod-dependent ribosome binding sites on endoplasmic reticulum (32, 293). Subribosomal particle formation was also blocked in rat liver (193) and housefly ovaries (6). Aflatoxin B Metabolism Species Comparison. Aflatoxin B metabolism was examined in a variety of species and several metabolites were identified (Table 1). There were striking differences between in vivo and in vitro metabolism and the metabolites found among species. found two significant differences in mice. 14 CB Wogan (264, 298, 300) metabolism in rats and First, the mouse excreted totally more toxin than the rat (8 9. 9% vs.. 80. 1%). The reduced urinary excretion of 14 ?.C by the rat accounted for the difference. Second, the rat retained more toxin in the liver than the mouse (7. 57% vs. 1. 51%). As indicated in Table 1, both the rat and the mouse form M, and B^ in vitro but M, was reported only 1 2a1 11 Table 1. Species Comparison of B Metabolites. Species Rat Mouse M. in vivo 1, 48, 71, 212, 236, 237, 267, 274 71 in vitro 27, 109, 212, 214, 231, 232, 258, 259 259 274 in vivo in vitro 215, 231, 232, 274 in vivo 67, 69, 70, 267 in vitro 185 Golden hamster in vitro 25 Monkey Guinea pig in vivo 212^ 267 in vitro 25, 212, 215 Rabbit in vitro Cow in vivo 72,.. 184 Calf in vitro 212b Sheep in vivo in vitro 25 Dog in vitro 25 Chicken in vivo in vitro Duckling in vitro Numbers refer to references. Conjugated metabolite. 185 215 216, 217 25, 212 212 Chick 67? 69? 70b 7, 141, 184., 196 in vivo in vitro 215 25 in vitro Goat 2a Metabolites R 175? 176b • 216 215 215 213 216 213, 216, 217 12 once from mice in vivo. As reviewed earlier, female rats and both sexes of mice were more resistant to B The mouse absorbed B (274). toxicity and carcinogenicity. more quickly from the stomach than the rat Female rats absorbed the same (237) or slightly less toxin from their stonaachs than males. [They.metabolized it toM^ at.the same rate as castrated males and much faster than normal males (236). liver cells in tissue slices took up B Mouse more slowly and metabolized it faster (231, 232). The rate of B metabolism was correlated with the basal aniline hydroxylase level for chick, guinea pig, calf, sheep, goat and mouse, but not for duckling, pig and rat. There was no trend in sheep, goat, rat, mouse and chick, which were more resistant to B toxicity, towards a higher microsomal hydroxylase level or greater rates of metabolism (212). B like the mouse. The highly sensitive duckling rapidly metabolized In contrast to the mouse, the major identified product of the duckling was aflatoxicol (R ) not M (26, 211, 218), R was formed by the 105, 000 x g supernatant and not by the microsomal pellet which produced M in the mouse, although both transformations required NADPH (210). The pattern of metabolism in the monkey varied dramatically w ith route of in vivo administration of tion with liver homogenates. 14. CB Aflatoxin P and upon in vitro incuba- as the glucuronide was the major urinary metabolite, 20% of dose, after i. p. injection and M 13 represented only 2. 3% of the dose (69). 14 CB , M After oral administration of accounted for 20% of the label and P , 5% of the dose, as the glucuronide and sulfate (67). Four days after i. p. injection 5. 6% was retained in the liver bound to proteins and an appreciable quantity bound to serum albumin. The urine and blood of monkeys retained detectable activity five weeks after oral toxin administration. sharp contrast, no P 30-50% of the B was found after in vitro incubation. was converted to Q In rainbow trout 50% of the in the urine in 12 hours. 14 and about 1%, to M CB In Instead, (68, 185). administered i. p. was excreted Half of that was unaltered B . The remainder was removed through the gall bladder into the lower gut or became bound to the nuclear and microsomal fractions of the liver (16). The LD50 for M. was 16 fig/40 g duckling compared to 12 (ig/ animal for B (235). It was carcinogenic to rainbow trout (268). had anti-microbial activity comparable to B , but B B_ was 200 times as toxic using the duckling bile duct hyperplasia assay (170). Accord- ing to the same assay, aflatoxiqol from fungal reduction of B was 18 times less toxic than B (76, 77). found from the reduction of B Diastereomers of aflatoxicol were by fungi (62). It was not presented whether they were separated for the duckling assay. No significant change in embryo viability or teratogenic effect was attributed to Pj uqder conditions yielding a positive chick embryo assay, with B, (275). At 14 doses of P 10 to 20.times greater than the B LD50 of 9. 50 mg/kg, only two mortalities: were observed in young mice (41). Activation. Several types of evidence have been reported which suggest aflatoxin B must be metabolized, that is, activated, to exert certain biological changes. Hypophysectomized rats fed 4 (jig B /g diet developed no liver tumors as compared to a 100% incidence in intact animals (1Z2). A marked inhibition of RNA synthesis was observed with intact rats receiving dietary B, but was not evident after hypophysectomy. The surgery or SKF 525-A decreased in vitro, micros omal drug activity resulting in an increase of a less polar metabolite of B produced by the 105, 000 x g supernatant and a decrease of the microsomally produced polar metabolite. Without surgery there was a decrease in the less polar metabolite which was further metabolized microsomally (108). Increased cytotoxicity of B to cell cultures was observed for those cell lines, including liver, able to metabolize the toxin (256). It was not shown whether the inhibitory product was a metabolite of B or arose from the cells themselves as a result of exposure to B . 7, 8-benzoflavone, a competitive inhibitor of micros omal oxidation, protected against the cytotoxicity of B and a host of other potential carcinogens suspected of requiring activation (262). Similarly, malignant transformation of cell cultures was demonstrated for B But 15 and numerous other carcinogens preferentially in cell lines capable of microsomal oxidation (295). As presented earlier in this review DNA template activity, RNA synthesis and DNA dependent RNA polymerase were inhibited in vivo but not in vitro. Moule and Frayssinet (195) were able to inhibit RNA polymerase in vitro with B in the presence of an active microsomal oxidative system or the extractible microsomal metabolites of B exclusive of M . Neal (199, 200) did not observe an inhibition of the polymerase but a decreased template activity. Template activity was reduced when DNA was present with a microsomal preparation actively metabolizing B . After removal of the microsomal system no inhibi- tory activity was observed in the remaining supernatant. The meta- bolite was either unstable or lost in the removal of the microsomes. Butler and Neal (49) proposed that the significance for carcinogenicity of B activation could be assessed with animals on a marginally choline-deficient diet which was protective against B toxicity but not RNA synthesis inhibition or tumor induction. Garner and others (114, 115) found male and female rat, guinea pig, mouse, hamster and human liver microsomes converted B to a reactive derivative toxic to Salmonella typhimurium TA 1530 and TA 15 31. B activation was dependent on oxygen and NADPH and inhibited by aniline, SKF 525-A and carbon monoxide. Microbial strains C207 and G46 were insensitive, presumably because they had 16 a viable DNA excision repair system which the sensitive strains lacked. Aflatoxins M , R and P. produced derivatives of lesser toxicity and B_ and B_ were not transformed. 2 2a No active metabolite was isolated. The common structure-activity relationship of the aflatoxins tested was the terminal double bond of the furofuran moiety. They proposed, as Schoental (261) had, that the active derivative was a highly reactive and unstable epoxide of the furofuran moiety. Xenobiotlc Metabolism by Fish Xenoblotics include all substances foreign to living organisms (87). They are generally lipophilic, facilitating entry to the organism across lipoidal membranes. To be removed from the organism they are metabolized to more hydrophilic compounds. They are meta- bolized by enzymes located primarily in the smooth endoplasmic reticulum referred to as microsomes after isolation, enzymes of intermediary metabolism in the cytoplasm called soluble fraction on isolation.and enzymes in the mitochondria. Microsomal reactions include oxidations, such as hydroxylation and epoxidation, and reductions of nitro and azo groups. tion alsp occurs. Nonmicrosomal oxidation and reduc- After metabolism by the above phase I reactions many xenobiotics are made more hydrophilic by conjugation to polar compounds such as glucuronic acid and glutathione (105, 209). 17 Gaudette, Maickel and Brodie (117) and Brodie and Maickel (36) reported fish lack the microsomal enzymes for xenobiotic metabolism. They suggested fish disposed of lipoidal compounds by direct diffusion through the gills and skin without initial metabolism. Baker, Struempler and Chaykin (20) reported that trimethylamine was microsomally oxidized. Since then microsomal metabolism has been reported for hydroxylation of biphenyl (64), 2-acetylaminofluorene (173), acetanilide and aniline (44, 81, 82), O-dealkylation of alkoxy biphenyls (63) and phenacetin (44), epoxidation of aldrin (53, 290), desulfuration of parathion (233), N-demethylation of anainopyrine (44, 81, 84), azo reduction (4), numerous nitro reductions (4, 43, 45, 139) and glucuronide formation (82, 95), The comparative metabolism between fish species was reported by Adamson (2), Dewaide (82), and Smith (271). Microsomal oxidation requiring NADPH and molecular oxygen was defined as a naixed function oxidase (182). The system was comprised of FAD-containing flavoprotein, NADPH cytochrorne c reductase, NADPH cytochrorne P-450 reductase and cytochrorne P-450 (181). b 5 Associated with it was another flavoprotein, iNADH. cytochrorne reductase and cytochrorne bc (35, 100, 120). b The same compo- nents were found for fish microsomes as other vertebrate systems (44, 45, 82). 18 Several conditions for measurement of in vitro microsomal oxidation were markedly different for fish (2, 44, 82, 83, 85). Little if any activity was found at the mammalian preparation incubation temperature of 37 Maickel (36). mately 25 in agreement with the earlier report of Brodie and Instead the optima were, for temperature, approxi- , , and for pH, near 8. 0. Higher NADPH levels and shorter incubation times were required. Dewaide (82) (see Table 2) found for p-hydroxylation of aniline the activity on a mg liver protein basis was higher for one fish species, roach, than for rat. Buhler and Rasmussen (44) reported the K m from Linewea-ver-Burk plots approximated mammalian species although Dewaide (82) always found greater values of K . m Dewaide (82) concluded that fish have an active microsomal system. Previous lack of evidence was due to using the same con- ditions,-, particularly temperature, for fish as for mammals. He argued that the contention of Brodie and Reid (37) that lipophilic xenobiotics do not require metabolism because they readily diffused through the lipoidal menabranes and skin was not a fact. Further, the consequences of metabolism also favored aquatic animals because water-soluble products would be restricted to the extracellular phase and in this way would be available in higher concentration for excretion through the gill, kidney and skin. Table 2. Comparative Micros omal Activity. Species a N-demethylation of aminopyrine /p-hydroxylation of aniline activity per g fresh liver mg liver protein mg liver DNA 100 g body wt. Mouse (Mus musculus) 19 /5. 8 0.11 /0. 034 9.0 /2. 7 90. /28 Rat (Rattus norvegicus) 15 /l. 3 0.09 /0. 008 7.4 /0. 65 59 / 5. 0 Roach (Leuciscus rutilas) 4.81/1.23 0.042/0.0103 2.17/0.52 13.2/3.40 Rainbow trout (Salmo irideus) l;10/0.35 0.009/0.0028 0.61/0.19 1.7/0.53 a From Dewaide (82, Table 17). vO 20 Chemical Initiation of Cancer Farber (101) summarized two current hypotheses: (1) the chemical, or a derivative, reacts with a cellular target inducing cancer directly, and (2) the process of initiation, through interaction of the chemical with cellular metabolism, induces an altered but not neoplastic cell that evolves into cancer. The Millers (190, 191) proposed that most chemicals needed to be metabolized to strong electrophilic reactants, the active products, to initiate cancer. Boyland (34) hypothesized epoxLdes were the active forms of polycycllc aromatic hydrocarbons. Dipple, Lawley and Brookes (90) presented the theory that the activity of the carcinogen and the site of action correlated with the stability of the epoxide. The hypothesis was extended by Jerina jt al.; (143) to other classes of compounds. As alternatives, activated methyl groups (90), free radicals (197) and others (291) were postulated as the activated or ultimate carcinpgen. Evidence for the possible biological significance of activated compounds included covalent binding of the synthetic compound to DNA (126, 241) and protein in vitro (23, 126); DNA, RNA and protein of cell cultures (125); DNA of mouse skin (51, 91, 242); and histones of rat liver (144). In the presence of hamster liver microsomes the epoxide of benzo[a]pyrene was formed and bound to DNA (28 9). Inhibition of aryl hydrocarbon hydroxylase prevented the 21 binding of the carcinogenic species to DNA, RNA and protein, and tumor formation in mouse skin (147). The transformation products of epoxides, phenols and dihydrodiols,were not active (204, 279). Farber (101) stated that DNA was often cited as the target because (1) activated carcinogens bound to it, (2) it. correlated to mutagenicity, (3) it was not turned over during the latent period before neoplasia first appears, and (4) it was the simplest hypothesis and did not require a complex of interlocking events. For mammals DNA alterations have also included single and double strand breaks (165, 166). With the latter the frequency of repair was dramatically decreased (101). Lijinsky et al. ,(168) and Baird et al. (19) have reported that all atternpts have failed to find the same products of the active carcinogen and DNA in vivo or in transformed cell cultures that were found with in vitro incubation of DNA. It was suggested that the number of interactions with DNA necessary for transformation was below the methods of chemical detection employed.. Consistent with this interpretation was the observation that the native DNA structure promoted a specific reaction with the amino groups of guanine which lay in the narrow groove of the DNA helix (241). Alternatively, changes in levels of glutathione, regulatory proteins (102, 103), NADH and NADPH (54), for example, have been reported to explain the initiation of cancer through metabolism of chemicals. 22 Farber (101) proposed that hyperplasia and cytotoxicity were required, for neoplastic change. Cytotoxlcity would promote the evolution (selection) of neoplastic cells. Through a series of experiments with cell cultures, Heidelberger (137) found that (1) individual cells were directly transformed by activated carcinogens as opposed to selection of pre-existing neoplastic cells, (2) transformation did not require the presence of a viral oncogene and (3) after activation most carcinogens were mutagens. Carcinogens as Mutagens Activated carcinogens were m.utagenic to mammalian cell cultures (22, 38), Neurospora crassa (161), Salmonella typhimurium (10) and Bacillus subtilis (178, 230). Ames, Lee and Durston (9) developed a series of tester strains of Salmonella typhimurium to assess the mutagenicity of potential carcinogens. Carcinogens, which failed to demonstrate mutagenicity themselves, were activated in the presence of an active mammalian mixed function oxidase (11, 12, 65, 66, 177) to mutagens. Table 3 illustrates the correlation among carcinogenicity, microbial mutagenicity and cell culture malignant transformation for chemicals in the presence of a viable activation system. Lethality to S^. typhimurium (114, 180) and B. subtilis (94) was found to correlate with the carcinogenicity of activated compounds in the same manner as mutagenicity in the same strains. Table 3. Carcinogens as Mutagens (11, 107, 192, 230, 244, 295). Compound for activation Carcinogenicity Microbial mutagenicity Cell culture trans formation 2-aminoanthracene 2 - am inof luor e ne 2-acetylaminofluorene benzidine 4-aminobiphenyl + + + + + + + + + + 4-amino-trans-stilbene 4 - dimethylamino-trans-stilbene p-(phenylazo)-aniline 4-(o-tolylazo)-o-toluidine NpN-dimethyl-p- (m-tolylazo)-aniline; + + + + + + + + + + 2 - naphthylamine 1-aminopyrene 6-aminochrysene benzofajpyrene 3-n^ethylcholanthrene + + + + + + + + + + + + 7, 12-dimethyl-benz [a]anthracene aflatoxin B i sterigmatocystin dinaethylnitrosamine N-methyl-N'-nitro-N-nitrosoguanidine + + + + + + + + + + + + + + + OJ 24 Ames (11) proposed that all the compounds tested acted through intercalation in the DNA base-pair stack since they were planar and, after activation, were franaeshift mutagen? and had an active side group that reacted covalently to DNA. pairing in bases of repetitive sequence. This would stabilize a shifted During DNA replication or repair this would result in an addition or deletion of base pairs in the DNA sequence. 25 EXPERIMENTAL Metabolism Studies Rainbow trout (Salmo gairdneri), Mt. Shasta strain, used for liver preparations were kept in fiber glass tanks with a water flow rate of 4 gal/min at a temperature of 12 9. 5 ppm. with an oxygen content of 8. 5 to Beginning two weeks before livers were taken, the fish were fed one of the diets (Appendix XI) to satiety at the same time of day seven days a week. Some were starved an additional two weeks after the same intensive feeding regime. Centrifugatjon Fractions of Liver Homogenates The trout were stunned, the liver quickly excised and perfused with ice-cold Krebs solution adjusted for fish (Appendix III). The fish weight, liver weight, sex and volume of bile were recorded. All sub- sequent operations were conducted in a cold room at 2-4 . Centrifuga- tion fractions were prepared from a resuspended acetone powder of liver or a whole liver homogenate according to Figures 2 and 3. If an acetone power was used for centrifugation, it was prepared (52) by homogenizing each g of liver in 10 volumes of -15 blender. funnel. dry acetone in a A precipitate was collected on filter paper in a Buchner It was resuspended in 10 volumes of -15 dry acetone and Kenmore Blender Model 600, Sears, Roebuck and Co. , Chicago, Illinois. 26 g whole liver or 0. 19 g.acetone powder 2 v phosphate buffer (Appendix III) homogenize 20,000 x g for 10 min supernatant supernatant 105,000 x gfor 90 njin 20, 000 x g for IS min supernatant 1 v to 10 v 105,000 x g for 90 min supernatant top layer microsomal pellet bottom layer microsomal pellet rinse and resuspend in v equal to original 10 v supernatant i I rinse and resuspend in v equal to original 10 v supernatant 105,000 x g for 90 min 105,000 x g for 90 min i ^" supernatant Figure 2. \layer top microsomal pellet supernatant Flow diagram of liver fractionation by centrifugation. bottom layer microsomal pellet 27 g whole liver 2 volumes 0. 25 M sucrose homogenize 12, 000 x g for 10 min i decant supernatant and measure volume I add 4 x v of 0. 0125 M sucrose with 8 mM CaCl pH 7. 2 stir a few seconds 600 x g for 10 min ++ k Ca precipitated microsomal pellet Figure 3. ••j supernatant Ca.++ precipitation of liver microsomes (145). 28 collected as before. The precipitate was washed with 10 volumes of peroxide-free diethyl ether, placed in an evaporating dish and dried overnight in a vacuum desiccator. The dry powder was ground to a fine powder with a mortar and pestle before resuspension in phosphate buffer. For whole liver preparations the liver was homogenized with 12 up-and-down strokes in a glass Potter-Elvehjem homogenizer tube and a loosely fitting teflon pestle. Preparation of Incubation Mixtures Lethal factor production was determined with one of the preparations shown in Figure 4. cate controls, All incubations were in duplicate with dupli- Each experiment was repeated at least once. As an alternative to direct addition of the toxin to the incubation mixture, trout were removed from their tanks, quickly stunned leaving the heart to continue functioning, and placed, ventral side up on a V-shaped restraining board. The peritoneal cavity was opened and the toxin was infused into the hepatic portal vein for one min and circulation to the liver was continued an additional 4 min. and perfused with Krebs solution for fish. assay are given in Appendix I, The liver was excised The details of the microbial The method of partial characterization of the phenotype of the strain of B_. subtjlis used appears in Appendix II. Neotetrazolium reductase activity and aldrin epoxidation were 29 3 ml of desired liver centrifugation fraction (CF) or control media (CM) in 25 ml Erlenmeyer flask or 1 ml CF or CM + 1 ml NADPH generating system + 1 ml MgCl2 (Appendix IV) ■</ 0. 1 ml DMSO, toxicant (Appendix V) in DMSO, toxicant in acetone (evaporate, add 0. 1 ml phosphate buffer), and/or piperonyl butoxide or cytosine or cysteine or lysine or glutathione (sometimes pre incubated before toxin addition for 5 min at 25° in shaker bath) (standard method: incubate 10 min, 25 in shaker bath) add 0. 1 ml J}. subtilis (Appendix I) incubate (standard method: 20 min, 25 Figure 4. in shaker bath) Incubation naixtures. 30 determined according to the methods In Appendices IX and X. Protein concentration was determined by the method of Lowry (174). Calculation of Microbial Viability The number of microorganisms for each treatment was determined from the triplicate plate count. The two methods used to express the results are illustrated in Figure 5. I, no. viable bacteria .nn — ' ''(treated) — ' — ■' x 100 no. viable bacteria (control) no. viable bacteria II. % reduction in viability = [l - Figure 5. (treated)—_ j no. viable bacteria (control) 100% Calculation of microbial viability. Isolation and Identification of Aflatoxicol Aflatoxicol from Trout Preparations Aflatoxin B was incubated with a 13, 500 x g supernatant plus 0. 5 M glucose or a 20, 000 x g or 105, 000 x g supernatant plus an NADPH generating system in Erlenmeyer flasks with an air atmosphere for 30 min to 1 hr at 25 on a shaking water bath. tion 1. 5-2. 5 volumes of chloroform were added. was drawn off in a separatory funnel. At the end of incubaThe chloroform layer The incubation medium was 31 re-extracted with 1. 5-2. 5 volumes of chloroform:water (3:1). Emulsions were broken by centrifugation at 2, 000 rpm for a few minutes. The chloroform extract was dried over Na_SO.. 2 4 Aflatoxicol was isolated by repeated preparative TLC on MN-silica gel G-HR 2 with benzene:acetone:ethyl acetate (50:6:12) as the solvent or by chromatography on silica gel H 3 columns eluted stepwise with hexane, hexane:diethyl ether (7:3), (6:4) and (1:1). The remaining aflatoxin B and unknown metabolites were removed with chloroform:acetone: isopropanol (87:10:3). The column fractions containing aflatoxicol were located by TLC with MN-silica gel G-HR developed with benzene; acetone:ethyl acetate (50:6:12) or chloroform:acetone:isopropanol (87:10:3). Aflatoxicol Diastereomers from Chemical Reduction of B B was chemically reduced to the diastereomers of aflatoxicol by the method of Pawlowski (219)and purified by.column'chroma.tography with neutral alumina (Appendix VII). The diastereomers as well as R o from fish were identified by UV and mass spectra (Appendices V and VIII). 2 3 Brinkman Instruments, Inc. , Westbury, New York. Brinkman Instruments, Inc. , Westbury, New York. 32 Extraction, Purification and Identification of M-.A, B0 A and NM 1 2a After incubation, the preparation, containing an appropriately treated centrifugation fraction of trout liver, toxicant and/or other compounds (Figure 4) in a 24 ml Erlenmeyer flask was transferred to a 250 ml Erlenmeyer flask. The smaller flask was rinsed with 96 ml of chloroform:acetone (58:38 or 38:58) and the rinse was added to the 3. 2 ml of media in the 250 ml flask. shaking on a water bath for 1 hr. at -2 3 . Extraction was continued by The extracts were stored overnight The volume of extract was reduced on a flash evaporator and re-extracted with hexane. The remaining fraction was re-extracted with either acetone:chloroform:water (58:38:4 or 38:58:4), acetone or chloroform. The amount of label in the extracts was determined by scintillation counting (Appendix XII). Fluorescent metabolites were observed by exposure to 365 nm UV light on silica gel TLC plates (Appendix VI). Purification of MA, B9 A and NM was first tried by preparative TLC on MN-silica gel G-HR developed with chloroforrruacetone (3:1) and column chromatography with silica gel 60 4 eluted with benzene:acetone: ethyl acetate (50:6:2) followed by stripping with acetone. 4 Separation of the metabolites was also attempted by high Brinkman Instruments, Inc. , Westbury, New York. 33 5 speed liquid chromatography on a 2.1 x 500 mm stainless steel column packed with Vydac and eluted with chloroform. Crude separation was achieved on 5 g of silica gel H packed with hexane on a 30 x 300 mm column. 7 The column was washed with hexane and the sample applied in acetone or chloroform. Lipid soluble material was removed with hexane and hexane:diethyl ether (50:50). The compounds were separated with sequential 50 ml addi- tions of chloroform, chloroform: isopropanol (99:1, 98:2, 97:3, 96:4) eluted with approximately 0. 5 psig nitrogen. The fractions containing predominantly B Li a. A and NM were separated on 8 g of activated silica gel H (110 for 2 hr) in a 20 x 500 g mm column. The compounds were eluted with a gradient of chloroform to chloroform:isopropanol (8:2) under pressure with a metering pump 9 at 60 ml per hr. Final purification of M A, B_ A and NM was accomplished by X Ct a. repeated chromatography on 4 g silica gel (0. 05-0. 2 mm) 5 in a Model 3100, Chromatronix, Inc., Berkeley, California. Chromatronix, Inc. , Berkeley, California. 7 o 9 Chromaflex column K-420540, Kontes Glass Co. , Vineland, New Jersey. Jacketed Chromaflex extender, K-422430, Kontes Glass Co. , Vineland, New Jersey. Cheminert metering pump, Model CMP-1, Chromatronix, Inc., Berkeley, California. Brinkman Instruments, Inc. , Westbury, New York. 34 30 x 300 mm column. The support was washed with 25 ml chloroform, an increasing gradient of 50 ml chloroform to chloroform: isopropanol (50:50) followed by decreasing the gradient over 50 ml to chloroform, and 50 ml chloroform. The samples were applied in 0. 25 ml chloro- form followed by five 0. 25 ml washes of the sample container. Frac- tions were collected with 100-300 ml chloroform and, where necessary, followed by 1% increases in isopropanol every 50 ml. After each column the fractions containing metabolites were located by their fluorescence with 365 nm UV light on TLC plates of MN-silica gel G-HR developed with chloroform:-acetone:isopropanol (87:10:3) and benzene:acetone:ethyl acetate (50:6:12). Non-fluorescing material was observed by iodine-staining, coumarin spray (Appendix VI), 254 nm UV light absorbance on MN-silica gel G-HR F..,, or by ^54 12 locating the label with a radiochromatogram scanner. Similar fractions were combined and concentrated on a flash evaporator. UV and mass spectra were taken. Acute Toxicity Trial Nine-month-old rainbow trout, fasted 48 hr and weighing approximately 60 g, were given a single intraperitoneal injection of Brinkman Instruments, Inc. , Westbury, New York. 12 Varian Aerograph, Walnut Creek, California. 35 the toxicants. Immediately before injection, each fish was anesthetized with MS-222 (tricaine methane sulfonate 13 ) and weighed. They were injected at a point just anterior and dorsal to the left pelvic fin. toxicants were dissolved in distilled dimethyl formamide (DMF administered in a volume of 1 jil DMF/g of body weight. are indicated in Table 15. (p. Aflatoxin B 14 The ) and Dose levels 66). was produced by Aapergillus flavus (ATCC 15517) cultures grown on rice, isolated and purified according to the method of Ayres (16). The diastereomers of aflatoxicol were prepared as described earlier. The feeding response was determined by presenting a small amount of diet each day to the fish. At the same time their physical appearance and behavior was observed. Mortalities during the 10-day trial were recorded and the fish autopsied.. At the end of day 10, the surviving fish were sacrificed. The LD50 was determined by the metho'd of Litchfield and Wilcoxon (171). During the autopsies gross abnormalities were recorded and the livers and kidneys were removed and preserved in Bouin's fixative. These tissues were sectioned at 4 |j. with a microtome and stained with hematoxylin and eosin for light microscope examination. 13 14 Sando, Inc. , Hanover. New Jersey. Merck Co. , Inc. , Rahway, New Jersey. 36 Chronic Toxicity and Carcinogenicity Trial The rainbow trout eggs were spawned and hatched in our laboratory facility and the fry held on control diet 3 (Appetidix XI)4 months before initiation of experimental diets. randomly selected. Groups of 120 fingerlings were Each group was held in a 6 gal plastic bucket, with many holes for water flow and an effective rearing area of 0. 5 cubic ft. Two buckets for duplicate diets were suspended in 4-ft diameter fiber glass tanks with a flow rate of 4 gal/min at a temperature of 12 with oxygen content of 8. 5-9. 5 ppm. After 2 months, each bucket of trout was transferred to an individual 4'-ft fiber glass tank. Duplicate groups of trout received diet 3 (Appendix XI) with the toxicants indicated in Table 16 (p. "78 ). One ml of ethanol/100 g of herring oil was used as the toxicant carrier and added to the control diets. After 4 and 8 months, 40 trout were taken from each tank. The trout were killed with MS-222, weighed and autopsied for gross abnormalities. The livers and kidneys were examined for tumors and toxic damage with a dissecting light microscope. They were preserved in Bouin's fixative and were1 prepared for histological evaluation in the same manner as the acute toxicity trial samples. 37 RESULTS AND DISCUSSION Microbial Assay The primary objective was to determine whether rainbow trout (Salmo gairdneri) activated aflatoxin B . Despite indirect evidence, the identity of the active metabolite from any species had not been reported. Explanations were that it was produced in extremely small quantity or was highly reactive and quickly bound to subcellular targets or was unstable. metabolite. A microbial assay could be used to detect a fleeting Bacillus subtilis GSY 1057 (hisAl, uvr-1, metB4), insensitive to high levels of B (30), was examined for the assay of the metabolite based on the hypothesis that it would be incapable of excising nucleic acid bases which had an active metabolite covalently bound and cell death would result. When cloned, the B. subtilis culture for this study required histidine and methionine and 99% lethality was observed in the presence of mitomycin C. This agreed with the phenotype (140). As shown in Table 4, there was amarked reduction in viability of microorganisms incubated with centrifugation fractions of liver which had been infused in vivo th.rough.the hepatic portal vein. some lethality from unaltered B Since with rich media had been reported (30), Pennassay broth was used as a control. The reduction was one-third of that found with a viable liver fraction. 38 Table 4. Lethal Factor Production. Fraction Liver (g)/incubation volume (ml) Phosphate buffer Treatment % Reduction (MB,) in viability10 3.5 20, 000 x g supernatant (boiled) 15/32 lo"4 7.0 20, 000 x g supernatant (boiled) 15/32 4 0. 3 x 10" 9.0 4 ID- 32. 6 15 /32 10-4a 99. 9 105, 000 x g pellet 1. 5/3.2 10-4a 30 20, 000 x g supernatant 1. 5/3.2 lo"4 99. 9 20, 000 x g supernatant 1. 5/3.2 4 0. 3 x ID" 99. 9 20, 000 x g supernatant 1. 5/3.2 lO"5 99. 9 Pennassay broth 105, 000 x g supernatant Intravenous infusion in vivo. 39 Incubation of B1 with the 20, 0.00..-X g supernatant gave the same reduction in viability as fractions from liver after in vivo infusion of B . Out of 10 7 cells, only 10 3 survived. Figures 6 and 7 indicate that a maximum response was approached with a 20 min microbial incubation and 10 -5 M B in the media. The same magnitude of effect occurred if acetone wds used as the solvent for B and was removed by evaporation before addition of other materials. A 0. 01 ml of ethanol as the solvent for B did not alter the assay. A 0. 1 ml of ethanol produced a 10% reduction in viability and reduced the magnitude of the lethality with B present. It was not determined whether the protection by ethanol was a direct inhibition or a generalized effect such as protein denaturation. It was decided that B. subtilis would serve as an effective assay. Enzymatic Conditions for Lethal Factor Production The next objective was to define the enzymatic conditions-l for lethal factor production. As seen in Table 5, the type of protein, level of protein and feed intake clearly influenced lethality, B induced hepatoma (153), cyclopropenoid fatty acid damage (277) and histologically observed liver changes (154, 155) paralleled the above results for corresponding diets. Restoration of some of the lethality by NADPH addition suggested a cofactor requirement. an enzymatic requirement for B activation. This supported The temperature 40 100 -® o o -H X ^ ^ (1) o H -M a 4-> In o o -M 10 ^-4 N-^- n) .^ M 0) -M o d .^4 (H (U 4-> CJ n) nJ Xi ^3 0) —^ ^-4 rH rO <U rt > > Q • o o c c OJ .-J .»-H 0. 1 Time (min) Figure 6. Loss of viability as a function of microbial incubation time. (20, 000 x g supernatant and 10"6 MB). O p P> <-r H P A o o p o Cd O p n P 1" U1 en CD o to O o o o no. viable bacteria (treated) x 100 no. viable bacteria (control) o o *>■ 42 Table 5. Effect of Diet and Feeding on Lethal Factor Production by 20, 000 x g Supernatant. a Liver wt. % body wt. Protein (mg)/ 3. 2 ml incubate % Reduction in viability Casein 1. 51 88.8 99 FPC1? high protein 1. 57 80.4 90 b FPC, low protein 1. 5 90. 5 79 Starved + NADPH 0. 65 48. 1 69 Starved 0. 65 48. 1 0 D let a Incubation media contained: homogenate from 1. 5 g liver/3. 2 ml and 1 0"6 M B Fish protein concentrate. 43 limitations and heat sensitivity of the system. (Table 6) agreed with previously reported conditions for active fish enzymes. In the early experiments little activity was. found in the 105, 000 x g pellet from homogenates of 1 g liver diluted two to three times. Activity was retained in the concentrated 10 5, 000 x g supernatant. Patterson and Roberts (216) had reported aflatoxicol was produced by the soluble enzymes in the supernatant and required NADPH. Pre>- liminary evidence for a metabolite with similar TLC characteristics had been found in this laboratory. Perhaps it was the lethal factor. For this reason, it was imperative to determine which fraction produced the lethal factor. It was possible that the lethal activity was associated with microsomes retained in the 105, 000 x g supernatant because the horn ogenate was too concentrated, therefore, retarding the sedirnentation. The supernatant would contain the necessary NADPH as found in a 20, 000 x g supernatant. At 10 MB, which gave more than 99% lethality with a fresh 20, 000 x g supernatant, no lethality was observed with a supernatant from an acetone powder extract of liver (Table 7). fold increase in B After a hundred- concentration, the lethality increased to the level seen earlier with a rich broth. The enzyme did not appear to be an acetone precipitable soluble protein, or it was inactivated by the method of preparation. After the liver homogenate was fractionated by centrifugation, neotetrazolium (NT) reduction and microbial death were used as 44 Table 6. Effect of Incubation Temperature and Prior Heat Treatment Incubation temperature of 20, 000 x g supernatant Protein (mg)/ 3. 2 ml incubate % Reduction in viability 25° 66. 9 99 30 95. 4 94 37 66. 9 0 b 77. 7 14 25° 159. 9 0 25 Incubation media contained: . homogenate from 1. 5 g liver/3. 2 ml; 1. 5-1. 9% liver wt/body wt; lO-6 M B . Supernatant heated at 80 for one min prior to addition of B . Same as b with twice concentration of liver. Table 7. Effect of 20, 000 x g Supernatant of Acetone Powder Preparation. Livera (g)/ 3. 2 ml . incubate Protein, (mg) / . i.3. 2 ml: incubate % Reduction in viability 1. 50 77.8 HT4 30.6 0. 75 38. 9 lO'4 28.0 38. 9 5 20. 5 0. 75 10' 6 1. 50 77.8 lO" 0 1.50b 77.8 10'6 0 1. 6% liver wt/body wt.. b Treatment (MBj) NADPH generating system added. a 45 markers of enzyme activity. Chan (52) found some NT reductase activity was dissociated from the 105, 000 x g microsomal pellet and occurred in the supernatant. The microsomes from fish had lower epoxidative activity than from rats. Chan's post microsomal fraction from fish produced a greater than additive enhancement of microsomal activity when incubated with rat microsomes. by Ca Microsomes prepared precipitation (21, 145) were reported to retain the soluble proteins lost by the above methods. As indicated in Tabled, Ca. NADPH-dependent NT activity. for microbial death. precipitated microsomes possessed Inconsistent results were obtained With insect microsomes (21), the Ca reduced cytochrome P-450 levels.. Reduced activity was reported (145) for rats because Ca++ and Mg sites. treatment competed for the same binding This could have been the case with the fish preparations. Dewaide (82) found fish required a greater Mg mammals. Sucrose was also used in the Ca concentration than precipitation prpcedure and has been reported to yield microsomes with reduced activity (187). The microsomal pellet from centrifugation was composed of a clear gel bottona layer and a top layer like a typical rat pellet. was true even from starved fish. This As seen in Table 9, rinsing and recentrifugation reduced the naicrobial activity of the bottpm layer, although protein and NT activity were still detectable. The specific 46 Table 8. Lethal Factor Production and NT Reductase Activity of 20, 000 x g Supernatant and Ca++ Precipitated Microsomes. Incubation conditions ProteirV(mg) / • 3; 2 ml iticubate NT reductase % Reduction in viability 20, 000 x g supernatant 88.8 76. 2 78 6. 69 ^ ++ • -^ i. , Ca precipitated microsomes No NADPH 15. 0 0 3 No;NADPH 22. 7 0 8 Low NADPH 22. 3 6. 17 37 High. NADPH 22. 7 1. 98 0 High NADPH 15. 0 8. 17 66 Incubation media contained: homogenate from 1. 5 g liver/3. 2 ml; 1. 9-2. 3% liver wt/body wt; lO"6 M B . 47 Table 9. Lethal Factor Production and NT Reductase Activity of 105, 000 x g Fractions. a Protein,'(rag) / :3. 2 mliricubate Incubation conditions NT reductase % Reduction in viability 105, 000 x g supernatant 98. 1 94. 2 Fen^ale Male 4. 9 8. 6 Bottom layer microsomal pellet 1. 5 1. 5 1, 36 NADPH Starve db + NADPH trace trace 0 11 88 0 Bottom layer microsomal pellet (rinsed) 1. 32 1. 14 Female + NADPH Male + NADPH 0. 76 0. 88 6. 3 0 Complete microsomal pellet 12.7 NADPH 7.23 61 Top layer microsomal pellet (rinsed) No NADPH NADPH NADPH NADPH NADPH NADPH 14. 3 14. 3 11. 1 12. 0 1.2C 0. 12d --9. 9 15.0 9.6 4.2 0 99 95 99 99 41 Incubation media contained; homogenate from 1. 5 g liver /3. 2 ml; 1, 5-1. 9% liver wt/body wt; 10"6 M Bj. 0. 63% liver wt/body wt. "Homogenate from 0. 15 g liver /3. 2 ml. Homogenate from 0. 015 g liver/3. 2 ml. 48 activity of NT reductase and microbial lethality markedly increased on a mg protein basis for the top layer compared to the post-mitrochondrial fraction and complete microsomes. Neither NT reductase nor micro- bial death was evident with boiled controls. Since adjustments were not made for variations in turbidity between fractions for the NT reductase assay they should be interpreted as trends and not compared as absolute values. The conversion of aldrin to dieldrin, a known microsomal epoxidation requiring cytochrome P-450 in fish (52), was examined in the post-mitochondrial fraction. Piperonyl butoxide (PB) presumably bound to cytochrome P-450 and inhibited epoxidation (132). incubation of PB with the preparations before aldrin or B "Without addition only a slight reduction occurred in epoxidation and none in microbial death (Table 10). After preincubation with higher levels of PB there was a marked inhibition of lethality, that is, an average of 2. 3 x 10 cells /ml out of 2. 3 x 10 cells /ml with B alone. 7 cells /ml survived compared to 1. 6 x 10 5 Epoxidation was inhibited to a much greater extent. Again, as the system was refined the specific activity increased as seen with the top layer. 8 x 10 3 x 10 5 3 In the presence of PB an average of cells/ml out of 2. 6 x 10 cells/ml with B alone. 7 cells/ml survived compared to The incomplete inhibition of lethal factor formation agreed with the results of Garner, Miller and 49 Table 10. Effect of Piperonyl Butoxide (PB) on Aldrin Epoxidation and Lethal Factor Production, a m Protein (mg)/; o -. . 1.3.-2 ml? iticuba-te ,. Treatment pmoles dieldriu/ „, _ , i-- • \ i ■ % Reduction mm mg ■ in viability proteiii) 20, 000 x g supernatant Aldrin 5 Aldrin + 10" 10" M PB M B 10"6 M B + 10"5 M PB Aldrin 3 Aldrin + 10" b M PB 10"6 M B 10~6 M B + 10^3 M PBb 79.2 2.79 79.2 2.59 79. 2 -- 99. 0 79.2 -- 99.0 88.8 3.04 88.8 0.12 88. 8 -- 98. 0 88.8 -- 74.0 Top layer microsomal pellet Aldrin 11.4 Aldrin + 10"3 M PB 11.4 0.65 11. 4 -- 97.0 11.4 -- 91.7 6 10" M B 10"6 M B 3. + 10"3 M PB Incubation media contained: 1. 6-1. 8% liver wt/body wt. 21.4 homogenate from 1. 5 g liver/3.2 ml; PB incubation before addition of aldrin or B . 50 Miller (114) that B rapid. conversion to the toxic product was extremely Aldrin epoxidation which was slower was inhibited 96%. Boil- ing completely inactivated the system. The electron micrographs (Figures 8 and 9) indicate the top microsomal layer was composed of vesicles of smooth endoplasnaic reticulum extremely free of other subcellular components. the data suggested that the microbial assay indicated B Together, conversion by an enzyme system associated with the snaooth endoplasnaic reticulum with properties of the NADPH-dependent mixed function oxidase reported for trout. Metabolism of Aflatoxin The purpose of establishing the microbial assay and the enzynaatic conditions which mediated microbial death was to identify new biologically active metabolites of B . Effect of Inhibitors The hypothesis was that the ultimate biologically active metabolite was unstable and had a short half life. epoxide of B (Figure 1). An example would be the As an electrophilic derivative it could readily react with nucleophilic compounds added to the incubation medium. It was postulated in this manner the covalently bound derivative of the ultimate metabolite could be trapped for isolation 51 m ,»■ * i » Figatie 8. <} Top layer 105, 000 x g pellet composed primarily of smooth endoplasmic reticulum. Electron micrograph of pellet fixed in 5% gluteraldehyde, stained with 1% OsO^ and section embedded in araldite-epon mixture. X41, 200. i 1 K' * . ,, Figure 9. V'r: , nm Same as Figure 8 (above). ■ 1 Ik X105, 000. 1 52 and identification. The microbial assay could be used to determine the success of a nucleophile as a trap. Microbial lethality would be less for better traps. In order to reduce the amount of lethality to a level that the effectiveness of a trap would be readily apparent, 20, 000 x g supernatant fraction from two-week starved fish were used. of lethal factor production 10 B MB was itself inhibitory but 10 gave the expected reduction (Table 11). 50 x 10 -5 -3 -3 -6 M At either level of B M cytosine reduced the lethal effect. tion of cytosine to 50 x 10 10 -5 At that level Raising the concentra- M produced lethality without B . Lysine, M, was an ineffective trap. Since the top portion of the microsomal pellet in the presence of an NADPH generating system was responsible for lethal factor production, a 1:100 dilution of it was tried. Cytosine at 10 not lethal but it reduced the lethality in the presence of B -3 M was by 60%. Increasing the enzyme concentration enhanced microbial death. Addition of cysteine, although somewhat toxic, reduced lethality by 50% while glutathione was ineffective even if soluble fraction was added back. Extraction of Metabolites The microbial assay, the enzymatic requirement and the effect of added nucleophiles were evidence for a metabolite(s) of B In 53 Table 11, Effect of Selected Nucleophiles on Lethal Factor Production. % Reduction in viability Treatment 20, 000 x g supernatant a 10"5 M B1 10"5 M Bj + 50 x 10 M cytosine 50 x 10"5 M cytosine 19. 7 0 0 20, 000 x g supernatant 6 10" M Bj 10"6 M Bj + 50 x 10" M cytosine 50 x 10"5 M cytosine 97 0 92.8 Top layer microsomal pellet lO"6 M B.1 _ -i 10"6 M Bl + 10 M cytosine 10"3 M cytosine 15 0 6 Top layer microsomal pellet 10"6 M B 10" M B + lO"6 M Bj + glutathione (reduced) glutathione (reduced) lO'3 M cysteine 10''3 M cysteine 64 16 58-67 20 29-31 Incubation media contained: homogenate fronn 1. 5 g starved liver/ 3.2 ml; 1.0% liver wt/body wt; 15 9 mg protein/3.2 ml. Incubation media contained: homogenate from 1. 5 g starved liver/ 3.2 ml; 1.0% liver wt/body wt ; 100 mg protein/3.2 ml. Incubation media contained: homogenate from 0. 015 g liver/3. 2 ml; 1. 5% liver wt /body wt; 0. 118 mg protein/3. 2 ml. Incubation media contained: homogenate from 0. 030 g liver/3. 2 ml; 1. 2% liver wt /body wt ; 0. 212 mg protein/3. 2 ml. 54 order to isolate the metabolite(s) various extraction procedures were examined. The results of extraction, after incubation of-various trout liver preparations, with chloroform:acetone:water (58:38:4 or 38:58:4) are given in Table 12. The designations of suspected metabolites were based on the proximity of R (TLC) plates to standards. 's on thin layer chromatography That is, suspected metabolites with an R_ equal to aflatoxicoj. were called R F and compounds with R 's F o F similar to B„ or M, were designated B0 A or M.A. e 2a 1 2a 1 Little R F was isolated from 105, 000 x g supernatant from o dilute homogenates of fed or starved fish. Appreciable R F was formed after addition of an NADPH generating system to the preparation of fed trout. Less was found from starved fish. A new metabolite, NM, was observed near the origin of TLC plates. Like all the metabolites, it fluoresced on exposure to UV light with a 365 nm wavelength. polar than M, and B_ . 1 2a In all TLC systenns used it was more The most NM occurred with the same condi- tions which produced maximum microbial lethality, Addition of two nucleophiles, cytosine and cysteine, decreased the amount of NM and microbial death. NM occurred. In parallel, a new fluorescent spot more polar than Addition of three times the amount of B as substrate or doubling the incubation time increased the yield of metabolites nonlinearly. « of the label After incubation with 14 C labeled B , 99+% w?as found in the metabolites and unaltered B . None of the suspected Table 12. Metabolites by TL.C. a Fraction Metabolites Incubation conditions 20, 000 x g supernatant (starved) 20, 000 x g supernatant (fed) RoF> BZaA 105, 000 x g supernatant (starved) + NADPH R0Fb 105, 000 x g supernatant (fed) + NADPH RoFb Top layer microsomal pellet (starved) + NADPH Glutathione (reduced) Cysteine Cytosine NM B 2aAC -o^' B2aA' NM B 2aAC' M AC I ' ^M^ NM tra P d NM , NM trap Top layer microsomal pellet (fed) + NADPH B2aA, MjA, NM ^CB,1 Cysteine 14 CR o Fd, 14 CB9^ia ,Ae, 14 CNMf NMd, NM trap Chloroforrruacetone (9:1); chloroform:acetone:isopropanol (87:10:3); benzene:acetonei ethyl acetate (50:6:2). R^F o fed>> R o F starved, < 1%, C e d f < 0.05%. i-5%. U1 3. 6-5. 3%. 56 metabolites were found after incubations with phosphate buffer, without NADPH and boiled fractions. Only a trace of R F was found from o 20,000 x g supernatant heated to 65-85 for 1 min. Apparently the soluble enzyme was more stable than the mtcrosomal system. Together, the evidence indicated that the TLC spots were enzymatic metabolites of B . The results suggested that R F was produced by an NADPHrequiring soluble enzyme similar to that reported by Patterson and Roberts (215). It did not appear to be the lethal factor since no micro-' bial death occurred after incubation of B fraction without microsomes. and the soluble enzyme B_ A, .MA and NM were postulated to 2a 1 be either the active metabolite or its degradation products, NM trap appeared to correspond to the postulated covalently bound products of the active metabolite and the added nucleophiles. Lethality and Identification of Metabolites i ' ' ' Y |i i— Preliminary evidence for several new metabolites was seen by TLC. Experiments followed to assess their microbial lethality and to identify the metabolites. Aflatoxicol was extracted initially from post-microchondrial preparations. It was isolated by preparative TLC. The UV spectrum in ethanol or methanol gave maxima at 331, 261 and 255 nm. The 57 maxima .and shape of the spectrum were in agreement with aflatoxicol from B transformed by Tetrahymena pyriformis (246, 283), Dactylium dendroides, Absidia repens, and Mucor griseo-cyanus (76, 77, 78), and Rhizopus arrhizus (62). The parent m/e 314 by mass spectrometry was in agreement with the mobecular weight. The presence of the hydroxyl was indicated by a peak at m/e 296 from the loss of a molecule of water. These and the additional peaks (Appendix VIII) were identical to those reported by other workers (62, 76, 246). After chemical reduction of B two compounds were obtained by preparative TLC or from column chromatography on alunaina. The one corresponding to the isolate from fish preparations moved ahead of the other in all TLC solvent systems employed and was designated F. Similarly, the one lagging in the rear was called R. Both gave UV and mass spectra identical to aflatoxicol and were designated R F o and R R. Cole, Kirksey and Blankenship (62) reported an identical pair of compounds from the degradation of B1 by Rhizopus arrhizus. Based on UV, infrared (IR), nuclear magnetic resonance (nmr) and mass spectrometry, they concluded the compounds were diastereomers. In addition they reported that during purification on silica gel in the presence of ethanol and acidic conditions the corresponding ethyl ethers of the diastereomers were found. For this reason, great care was taken during all isolations of R F and R R to avoid the use of o o alcohols and acids. A new column chromatography procedure 58 (Appendix VII) was developed to specifically circumvent artifact formation. With neutral alumina and benzene, ethyl acetate and acetone as solvents, no compounds were isolated that corresponded to the reported ethers. If R F or R R was- allowed to stand a day at o o ' room temperature iq. acidic methanol, compounds similar to the reported ethers were seen. They moved well ahead of B , R F and 1 o R R in all TLC systems examined, o Several extinction coefficients (« ) have been reported for R F o and one for R R (Appendix V). 14, 100. For R F they ranged from 13, 780 to They were similar to the decrease in c from 21, 800 to 13, 900 for the complete reduction of the cyclopentenone of B THDB,. 1 to Because the reported « of 25, 200 for R R was unexpected, r o a rough deternaination of the extinction coefficients was made. Enough pure R F and R R were not available for an accurate reevaluation. No difference was found. Further, no difference in fluorescence intensity on TLC plates was seen for a series of concentrations in duplicate based on 13, 780 as the extinction coefficients for both R F 0 and R R. o If the concentration of R R was based on 25, 200 as the o the fluorescence intensity of R R was greater than R F. 0 o o € The results suggested a value of 25, 200 provided too low an estimate of the concentration. For this reason 13, 780 was selected to compute the concentrations of R F and R R. o o , 59 R F without enzymatic activation did not produce a significant reduction in rnicrobial viability (Table 13). For some unknown reason the diastereomer, R R, not isolated from fish but produced by chemio ' cal reduction, repeatedly exhibited some toxicity. In the presence of 20,000 x g supernatant or purified microsomes with a NADPH generating system, R F was activated to a lethal product. o and Miller (114) reported similar results. Garner, Miller They found aflatoxins M , P and G which contain the vinyl ether were activated whereas no activity was associated with B_, G_ and B,, which lack the vinyl / 2 2 2a ' ether. The acetyl derivatives of M,1 and R O were prepared and no r t- correlation for a change in activity was found. In agreement with Garner's results B_ could not be activated but Q which has the vinyl ether was activated (Table 14). At concentra- tiong where a several log reduction in viability was observed with B and R F, less than a one log decrease was observed for Q . In this case the presence and location of the hydroxyl group was significant. A significant decrease in viability was not obtained with crude extracts of B, or R F incubation media, crude column fractions with 1 o labeled and fluorescent material and compounds B_ A, M,A and NM. 2a 1 Similarly, Garner, Miller and Miller (114) were unable to extract an active metabolite from rat, mouse, guinea pig, hamster and human microsomal preparations which in the presence of B factor lethal to Salmonella typhimurium TA 1530. produced a Neal (199) found, in 60 Table 13. Microbial Lethality of Unactivated and Activated Aflatoxicol Diastereomers. Fraction Phosphate buffer Phosphate buffer Phosphate buffer Phosphate buffer 20, 000 x g supernatant Top layer microsomal pellet + NADPH Table 14. Treatment 8. 9 x 10"5 M R F o 5 1. 8 x 10" M R F o 5 0. 7 x 10" M R F o 2. 9 x 10"5 M R R o 5 5. 0 x 10~ M R F o 0. 7 x 10'5 M R o F Comparative Microbial Lethality of B , Q Fraction Phosphate buffer Phosphate buffer Treatment 10"5 M B % Reduction in viability 0 6. 3 0 21-36 66 82 and B? % Reduction in viability 0 5 M Q] 0 5 ID' 20, 000 x g supernatant 10~ M Bj 99 20, 000 x g supernatant 10"4 M B- 0 Top layer microsomal pellet + NADPH 10"5 M B 99 Top layer microsomal pellet + NADPH lO"13 M Q 1 22 61 the presence of an active microsomal system and B , RNA polymerase was inhibited. If the polymerase was added after stopping microsomal activity, no inhibition was observed. preparation was not inhibitory. Likewise, an extract of the He suggested that inhibition was either dependent on some other microsomal function or that the metabolite was unstable or so reactive Lt was not extractible. In contrast, Sarasin and Moule (253) extracted an extremely polar compound from microsomal preparations of B which was inhibitory to the synthetic activity of normal rat liver polysomes in a cell free system. The seemingly conflicting results could be explained with the epoxide of B proposed by Schoental (261) and Garner, Miller and Miller (114). As a highly reactive electrophile it could react readily with nucleophiles such as DNA and RNA bases. Garner found micro- bial lethality was inhibited by addition of RNA and DNA. Only after reaction in the presence of active microsomes wasB, inseparable from RNA.by Sephadex G-'IO chTomatography. Alternatively, a diol could be formed by epoxide hydrase or nonenzymatically and react with nucleophiles. Instead, Gurtoo (127, 128) and Gurtoo and Dave (130) proposed the hemiacetal of B , B_ , was formed directly 1 or by rearrangement of the epoxide. <£a They obtained spectral evidence that in the presence of active microsomes, B, was bound as B-, to r 1 2a RNA and protein and could not be separated by Sephadex G-25 62 chromatography. Patterson and Roberts (213, 215) reported that the hemiacetal of B, readily opened. The aldehydes could bind to amino groups to give Schiff bases (Figure 10). o A diol could react similarly. o i^n-^nx^HO^o^-^-oC^ CHO CHOo*^OCH CH Aflatoxin B2a ^ CH -^-OCj, ^ T OOC E'R^'COO' Figure 10. Hypothetical Schiff base formation of amino acids with one of the resonance forms of the phenolate ion. Unfortunately, Gurtoo was not able to determine if the binding was a natural consequence of B_ metabolism or the result of chemical degradation in the presence of Tris-HCl. Since Garner and Gurtoo were attempting to identify the compounds bound to RNA, efforts as described below were made to isolate and identify B A, M.. A and NM directly rather than continuing with the trapped products. Purification by partition chromatography on cellulose or kieselguhr were unsuccessful. chromatography supports. The compounds absorbed to glass and To take advantage of this property, adsorption chromatography with various silica gels was employed. Attempts were rnade to avoid alcohols because aflatoxicol could be a precursor and it readily formed ethers with alcohols. Because the 63 compounds co-extracted and co-migrated with polar lipids and adsorbed so strongly to silica gel, it was necessary to use isopropanol to pretreat the columns. No alteration of R F on TLC indicative of ether formation was observed. After repeated column chromatography, fractions of each metabolite were obtained that were free of other UV fluorescing and absorbing material and I_-staining compounds^ The UV absorption maxima of B_ A were 263 nm and 220 nm. 2a NM had maxima at 261 nm and 255 nm like R F but had only an extremely minor absorption at 331 nm compared to R F. Koes, Forrester and Brown (148) extracted ametab- olite of B,, which displayed an absorption maximum at 270 nm, from active microsomes of guinea pig. They employed the same extraction pro- cedure as the present study; however, methanol was used to recover the metabolites from silica gel. Subsequent ether formation could explain the non-polar behavior of the metabolite. with authentic M MA co-migrated with chloroform:acetone; isopropanol (87:10:3) and benzene:acetone:ethyl acetate (50:6:12) as solvents. MA had a shoulder at 262 nm and maximum, absorption at 220 nm in the UV compared to 226 nm, 265 nm, 357 nm for M (141). Using mass spectrometry, no single m/e could be conclusively assigned as the parent peaks of any of the cornpounds. The compounds appeared to be much more labile than the standard B used to establish instrumental conditions. A characteristic spectrum was 64 obtained for B but the unknowns appeared to degrade into many fragments of low mass. Recently, Swenson, Miller ^nd Miller (280) reported 2, 3-dihydro2, 3-dihydroxy-aflatoxin B (B.-diol) from acid hydrolysis of a B derivative bound to rat liver rRNA. The structure was confirmed by comparison of the UV absorption in neutral and alkaline solution, the R 's in many TLC systems and mass spectra of the acetonide and r diacetyl derivatives of authentic synthetic material. The RT-.'S of NM were similar to those reported for B -diol in each of the solvent systems, Migration of NM and the reported B ^diol on H BO -silica gel sheets was retarded as expected for a vie-diol. The RNA-B adduct did not exhibit the bathochromic shift in alkali found with the B -diol and B_ . For this reason it was postu- lated the covaient link to RNA was through carbon 2. They speculated the major base attacked was the strong nucleophile guanine. (113) reported that the microsomal system bound B Garner to a greater extent to polyriboguanylic acid than any other homopolyribonucleotide. Determination of whether NM or one of the other metabolites is a similar diol would be extremely interesting siace Swenson only found the diol after acid hydrolysis of the B product bound to rRNA. 65 Acute Toxicity Trial Mortality and General Observations The mortalities due to B, or R F ten days after intraperitoneal 1 o injection are reported in Table 15. According to the method of Litchfield and Wilcoxon (171) the LD50 for B 95% confidence limits of 0. 35-0. 59 mg/kg. was 0. 46 mg/kg with The slope function was 1. 80 with 95% confidence limits of 0. 66-1.37. For R F the LD50 was o 0. 66 mg/kg with 95% confidence limits of 0. 46-0. 94 mg/kg. The slope function was 1. 78 with 95% confidence limits of 1. 19-2. 67. slopes for B and R F were examined for parallelism. The The slope function ratio was 1. 01 with 95% confidence limits of 0. 59-1. 75. Since the slopes were significantly parallel an estimate of the relative difference was made. of 0. 92-2, 22. potency. The ratio was 1. 43 with 95% confidence limits The compounds were not statistically different in No mortalities due to R R were observed. o Greater doses were not tried due to lack of additional pure R R. o The LD50 of B , 0,46 mg/kg, was less than the LD50 of 0. 81 mg/kg previously reported from this Laboratory {2/8). .Every attempt was made to duplicate the conditions of the original study and the explanation may be the variation in sensitivity of trout from eggs of different females. This variation was observed during trial experiments. Detroy and Hesseltine (76, 77) reported that R was 1/18 as toxic as 66 Table 15. Mortality in Rainbow Trout 10 Days after Intraperitoneal Administration of Aflatoxins B,, R F and R R. loo Total dose (me/ke bodv wt.) Mortality Aflatoxin B 0 (DMF control) 0/10 0.2 2/10 0. 3 1/10 0. 4 4/10 0. 6 6/10 0.8 9/10 Aflat oxin R F Aflatoxin R R 0 (DMF control) 0/10 0/10 0. 28 1/10 -7 T 0. 56 4/10 0/10 0. 85 5/10 — 1. 27 9/10 0/10 o 0 67 B. with the duckling bile duct hyperplasia assay. It was not clear whether they had separated the diastereomers of R o and, if they had, which one was assayed. The time interval between toxin administration and the first mortality was dose-dependent for both B and R F. doses of both, death first occurred on day four. mortalities were not observed until day eight. At the higher At the lower doses, For B dosed trout, inappetence, weakness and darkening in external coloration were observed a day or two before death. The time interval was only a few hours for trout given R F. o The severity of gross pathological changes of internal organs observed at autopsy paralleled the dose for both B, and R F. 1 o hemorrhaging appeared to be the cause of. death. pale patches at the lowest dose. Internal The liver had a few At mid* doses, it was almost entirely pale yellow to white in color and at high doses it was extremely mottled. The liver floated in Bouin's solution at the mid and high doses suggesting a decrease in density with increasing intoxication. The degree of multiple hemorrhages in the fat bodies and entire gastrointestinal tract increased with the dose. filled with blood at mid and high doses. visible internally at 0, 56 mg /kg R R. patches were observed on the liver. The intestine was No pathological cha,nges were At 1. 27 mg/kg only a few pale 68 Histological Observations Liver. The degree of liver damage paralleled the dose-response seen with mortality and gross pathology. A higher dose of R F was required to give the same degree of damage as with 0. 4 mg/kg B (Figures 11 and 12), R R produced significantly less damage and the dose-response was not the same (Figure 13). At all doses of B, and R F, altered liver architecture, parenchy1 o mal cell necrosis with cytoplasmic yaculation, irregular nuclei with vaculation and disruption of the vascular system with hemorrhaging were evident. In the case of 1.27 mg/kg R R (Figure 13), more normal liver architecture was retained as indicated by the prominent sinusoids and muralia, or cords, which were characteristically two cells thick. Gross pathological changes seen on atuopsy and damage seen histologically suggested R F was somewhat less potent than B despite the lack of statistical significance which was due to the large variation in response for the number of fish tested. Kidney. In the kidney, the degree of damage paralleled mortality and gross pathology for B (Figure 16), but not R F (Figure 18). Apparently less R R, 0. 56 mg/kg, was required to produce damage similar to 0. 85 mg/kg R F. o diastereomer R F. Trout converted Bn to the aflatoxicol 1 Perhaps the kidney was not capable of excreting 69 f** >>' "1* -» v - - ^ ■••.. . "A" Vte&: ^^ -.^ Figure 11. Liver from trout dosed with 0. 4 mg/kg B . Note the hemorrhaging and complete destruction of the cord structure and hepatocytes on the right. Some original tissue is seen on left.' Vacuoles are abundant and nuclei are irregular. Hematoxylin and eosin. X128. Figure 12. Liver from trout dosed with 0. 56 mg/kg R F, Generally similar to 0. 4 mg/kg B.. In upper left, tissue destruction is nearly complete. Nuclei are all bizarre. Bile duct in lower right appears unaffected. Hematoxylin and eosin. XI28. 70 Figure 13. Liver from trout dosed with 1. 27 mg/kg R0R with enlarged, vacuolated and bizarre parenchymal cell nuclei. Gross structure is essentially normal. Hematoxylin and eosin. X128, 71 Figure i4. Normal trout liver. The cords, or muralia, are two cells wide and separated by sinusoids in which Kupfer and circulating blood cells are found. Nuclei are uniform in size and shape. Note hepatic vein in upper left. Hematojcylin and eosin. X320. Figure 15. Normal trout kidney showing a cross section of a collecting duct (d), a glomerulus (g), and many sections of renal tubules, or nephrons, embedded in a matrix of hemapoietic tissue. Hematoxylin and eosin. X128.. 72 Figure 16. Kidney from trout dosed with 0, 4 mg/kg B . Note the enlarged, vacuolated and deformed epithelial nuclei in the tangential section of a renal tubule. Numerous vacuoles are present in the cytoplasm. Hematoxylin and eosin. X320.- Figure 17. Kidney from trout dosed with 1. 27 mg/kg R R showing the nuclei of the tangential sections of the second proximal segments of the tubules are greatly deformed compared to those in the first proximal segments. Hematoxylin and eosin. X320. 73 v> >m IOT ».^lm -^ Figure 18. Kidney from trout dosed with 0. 85 mg/kg R F. The only obvious sign of toxicity is the deformity of the epithelial nuclei of the nephrons. Hematoxylin and eosin. X128, Figure 19. Kidney from trout dosed with 0. 56 mg/kg R R. The nuclei show the deformities similar to those from trout receiving 0, 85 mg/kg R F (above). Hematoxylin and eosin. X128. 74 the unnatural diastereomer as well, hence the greater toxicity from R R. In all cases damage consisted of one or more tissue changes including necrosis, bizarre nuclei and abnormal glomeruli and hemapoietic tissue, Chronic Toxicity and Carcinogenicity Trial Each of the toxins was fed at a level of 20 ppb on the basis of the extinction coefficient (e ) reported in the literature (Appendix V). As discussed earlier, it was concluded that the e of 25, 200 for R R was o too high and that it more closely approximated 13, 780 for R F. With that e , R R was actually fed at 36. 6 ppb. Liver Chronic Toxicity. The form of toxic injury was similar, although less extensive, to that observed for acute administration. B and R F produced similar changes (Figures 20 and 21) and R R was much o o less toxic overall although severe in some cases (Figure 22), The addition of 50 ppm of cyclopropenoid fatty acids (CPFA) to the control diet (Figure 23) produced typical signs of injury (277). extensive regeneration was seen. In many fish The addition of CPFA to the B diets produced much regeneration with fibers present. This resulted in a reduction in apparent toxic damage compared to those not receiving CPFA. The response to R F and R R in the presence of CPFA 75 J Figure 20. Liver from trout fed 20 ppb Bj for four months with extensive parenchymal cell damage and abnormal nuclei, Note the islets of darkly stained, regenerating parenchymal cells. Blood vessel walls appear normal. Hematoxylin and eosin, X128, msttti .(< E»V-». .-i ' «\ »*••**% '£*j&tf' tte Figure 21. Liver from trout fed 20 ppb R-F for four months. Injury to the parenchymal cells is as great as with 20 ppb B (above) /e) although the extent of regeneration is less advanced. Hematoxylin and eosin. X128. 76 Figure 22. Liver from trout fed 36. 6 ppb R0R for four months. The extent of nuclear abnormalities in the parenchymal cells is nearly as great as with 20 ppb R F. There is no evidence of regeneration. Hematoxylin and eosin. X128. Figure 23. Liver from trout fed 50 ppm CPFA for four months. Many parenchymal cells contain fibers and are filled with lipid globules. The nuclei are generally normal. Hematoxylin and eosin. X128. 77 was extremely variable fronn normal.to severe damage with regeneration. No fibers were observed. Hepatorna. The incidence of hepatorna is presented in Table 16. Histological evidence for neoplasia is presented in Figures 24-27 for the four month sample. The incidence and severity of hepatorna paral- leled the degree of toxicity at four months with CPFA. were microscopic for R F and R R. o o Most nodes At eight months R R, the 0 o diastereomer not produced by trout, remained significantly less potent despite it being fed at nearly twice the level of B, and R F. 1 o It remains to be seen whether R R will be carcinogenic without CPFA at 12 months. If not, it would be interesting to know if it would at higher levels or, i,nstead, it acts as a cocarcinogen to CPFA, Kidney Chronic Toxicity. The kidney histology did not appear to follow exactly the same pattern among,: toxins although nuclear abnormalities were the primary symptom of toxicity. cantly increased tubule damage. Addition of CPFA signify Tubule damage was greatest with R R and nearly equal for B., and R F. o 1 o tNoneobliastic: changes were 0 observed in the kidneys. Significance of Metabolism of B Evidence has been presented that B, was metabolized to R F by i o Table 16. Incidence of Hepatoma at Four and Eight Months from Start of Feeding Trial. Hespatoma Incidence Diet Dupli.cate 4 mo. sample H epatoma/40 trout % Duplicate 8 mo. sample Hepatoma/40 trout % Control 0 0 0 0 Q Va v 0 0 0 18 27 56. 3 0 0 0 11 10 26. 2 b RoR 0 0 0 0 0 0 0 CPFA C 0 0 0 1 1 2. 5 Bj* + CPFAC 6 3 11. 2 39 38 96. 3 R0Fa + CPFAC 3 2 h. 3 38 37 93.8 R0Rb + CPFAC 2 1 3. 8 25 19 55.0 "20 ppb 36. 6 ppb '50 ppm oo 79 Figure 24, Liver from trout fed 20 ppb B. and 50 ppm CPFA for four months. The more basophilic tissue on the left is a portion of a hepatoma. Note that the nuclei are small, oval and uniform, and the cells contain no glycogen or lipid globules. The normal tissue on the right is filled with glycogen vacuoles and the nuclei are more irregular in shape and size. Hematoxylin and eosin, X128. Figure 25. Liver from trout fed 36. 6 ppb R R and 50 ppm CPFA for four months. Note the presence of a few large fluid-filled vacuoles and a few bizarre nuclei in the basophilic (hepatoma) tissue. The normal tissue (upper) contains many glycogen vacuoles. Hematoxylin and eosin. XI28. 80 Figure 26. Liver from trout fed 20 ppb R F and 50 ppm CPFA for four months. The small, superficial hepatoma is typical except for the vacuolated areas. The original contents of these areas is unknown. The scattered, basophilic areas in the normal tissue are regenerating parenchymal cells. Hematoxylin and eosin. X20, Figure 27. Detail of above photo showing edge of the hepatoma. Note the typical, small, basophilic hepatoma cells with their uniform, oval nuclei. Islets of regenerating tissue are seen in the normal liver (right). Hematoxylin and eosin. X128. 81 Ml [•^ ««• \ & Figure 28. Kidney from trout fed 20 ppb R0F for four months. Note the degenerating epithelial cells, bizarre nuclei and atypical cytoplasm. Hematoxylin and eosin. X320. i Figure 29. Kidney from trout fed 36. 6 ppb R R for four months. Much degeneration, cytoplasmic vacuoles and bizarre nuclei are evident in the second proximal tubules whereas the first proximal segments appear essentially normal. Hematoxylin and eosin. X320, 82 »l f> ^ ^ ^ Figure 30. Kidney from trout fed 50 ppm CPFA for four months. The abundance of vacuoles in the epithelial cells with essentially normal nuclei are early signs of tubular degeneration, Hematoxylin and eosin, X320, Figure 31. Kidney from trout fed 20 ppb Bj and 50 ppm CPFA for four months, A few of the trout such as the one above had degeneration of the epithelial cells with cytoplasmic degradation and bizarre nuclei, Hematoxylin and eosin. X320. 83 *** V M y> •^'^r Figure 32. Kidney from trout fed 20 ppb R F and 50 ppm CPFA for four months. The extent of damage among trout was variable. The epithelial cells of some had much cytoplasmic degeneration and extensive nuclear deformation as shown above. Hematoxylin and eosin. X320. '% f _. ^ r . _ % 9 <?•. Figure 33. Kidney from trout fed 36. 6 ppb R R and 50 ppm CPFA for four months. Extensive degeneration of kidney tubules was evident from R R fed trout. Note the bizarre nuclei and cytoplasmic vacuoles. Hematoxylin and eosin. X320, 84 soluble enzymes of the 105, 000 x g supernatant and NADPH. The microbial assay, in agreement with that of others (114), indicated B and R F were not toxic but were further metabolized to lethal products, o The metabolism was mediated by the microsomal fraction containing ^.n active NADPH mixed function oxidase. The data suggested the following pathways (Figure 34). microsomal ^7-r--,—, ' T A" ' WTT ' oxidase + NADPH T, B. 1 T, J.± -L. ,-J. B. active metabolite 1 soluble reductase + NADPH V R F o microsomal v —7T— , TVT A T-.T-.U ' pxidase 4- NADPH „ _, ,_■ . , ,-^ R F active metabolite o or II. B Figure 34. -* -) R F o Hypothetical pathways of B R F active metabolite o and R F metabolism. Pathway II is consistent with the evidence that B produce similar biological effects. and R F Pathway I reconciles the differ- ences in magnitude of activity between B and R F. It must be remembered that R F was administered extracellularly and the differo ence in degree of activity for similar doses may represent the difference in entry of R F into the cell. o Patterson and Roberts (213) found a TLC spot with an R^ corresponding to B, after incubation with R F F 1 o 85 and proposed R o F was metabolized back to B,11 and the B, was further metabolized. A similar spot was observed in this study but it did not have the UV or mass spectra of B . R F does not represent a significant detoxication of B whatever the pathway. Instead, R F conserves the presence of a potentially biologically active compound, bolite of the duck. by trout R F is also the major meta- This perhaps explains why these species are the most sensitive to B . Strong evidence has been reported that microsomal activation was required for both hepatic necrosis (243) and carcinogenicity (137) of aromatic hydrocarbons and aflatoxin. In both cases the first step was believed to be the transformation to the epoxide. The highly reactive electrophilic epoxide could covalently bind to critical cellular necleophiles or rearrange to toxic phenols. Farber (101) proposed that toxicity by killing many cells served as a selective pressure for the malignantly transformed cells. This would explain the comple- mentary consequences of microsomal activation of potential carcinogens and toxins. The validity of the hypotheses remains to be proven. It is possible the correlation of microsomal activity of toxicity and carcinogenicity is happenstance. Instead, the interaction of the carcinogens and toxins with naicrosomal oxidation may involve some yet unidentified process. Regardless, it is noteworthy that the trout, a 86 phylogeneticaUy more primitive species and the most sensitive one to B induced carcinoma, produced a lethal factor with an NADPH- dependent microsomal preparation similar to rodents and man. Areas for Future Study 1. Complete the identification of unknowns (metabolites). Chemical ionization spectrometry offers the potential of increasing the percent ionization of metabolites too unstable for conventional electron impact mass spectrometry. empirical formulas might be obtained. In this way the M. W. and Derivatives could also be made for conventional mass spectrometry. Repeated computer- mediated nmr scans of the small amount of products available could provide structural evidence. 2. Further characterize the enzymatic conditions for R F and other o metabolite fornaation. Perhaps in this way greater quantities of unknowns could be made and the metabolic relationships of the metabolites better understood. 3. Through the use of selected diets, inhibitors, age of trout or species, determine the significance of activation and the relationship of B, and R F to each other in the sequence of toxic and 1 o carcinogenic events. 4. Study the influence of R F and other metabolites on cellular o metabolism. 87 Determine what effect CPFA has on R F and metabolite producr o tion and their influence on metabolism. Determine the absolute configuration of R F and R R, for o o example with X-ray diffraction, and assess why the difference in structure is significant. 88 SUMMARY AND CONCLUSIONS Rainbow trout, Salmo gairdneri, Mt. Shasta strain, activated aflatoxin B., R F and Q. but not B0 to rproducts lethal to Bacillus 1 o 1 Z subtilis GSY 1057. Aflatoxicol, R F, was found as a metabolite. o It was formed apparently by an NADPH-dependent soluble enzyme of the 105, 000 x g supernatant from liver. Activation to products lethal to B. subtilis required the 105, 000 x g pellet and NADPH and was dependent on the diet. An active mixed function oxidase with neotetra- zolium reductase and aldrin epoxidase activity was present in the microsom.es. Activity was reduced with piperonyl butoxide, NADPH deprivation and heat treatment. After metabolism of 14 C labeled B the label was distributed between unaltered B and three extremely polar products designated NM (3. 6-5. 3%), B A (1-5%) and MA (<1%). Their presence paralleled microbial death and they were eliminated or reduced along with microbial lethality when cytosine or cysteine were added to the incubation media. The LD50 of the diastereomer of aflatoxicol (R F) formed by trout was 0. 66 mg/kg compared to 0. 46 mg/kg for B . There was no significant statistical difference in potency although gross and microscopic pathological changes were less for R F. o No lethality occurred and toxic injury was much less severe for the other diastereomer 89 (R R) produced by chemical reduction even at the highest levels of o R F tested, o After eight months of feeding 20 ppb of B and R F and 36. 6 ppb R R, the relative incidences of hepatoma were 56. 3, 26. 2 and 0% o respectively. The incidences with the addition of 50 ppnn CPFA were 96. 3, 93. 8 and 55% respectively. It was concluded that aflatoxicol was not an effective means of detoxication of B , but instead extended the presence of a potentially toxic and carcinogenic compound. It was suggested that enzymes present in trout microsomes activate aflatoxins B , aflatoxicol and Q to the ultimate toxins and carcinogens. the vinyl ether and B Since they each possess does not, it appeared that this structural feature was required for activation. 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APPENDICES 120 APPENDIX I MICROBIAL ASSAY Use aseptic technique for all preparations, including trout enzymes, that are in contact with the microbial assay. A. First Culture 1. Streak a fresh tryptose blood agar base 15 plate with Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1) and incubate 12 hr at 37 . 2. Add 20 ml Pennassay broth (PAB) 17 to 250 ml side arna culture flask. B. 3. Inoculate flask with streak off fresh 12 hr plate, 4. Incubate fla,sk at 37 on rotary shaker at Z50 rpm. Second Culture 1. Add 10 ml of PAB for each 3 ml of cells desired to a 250 ml side arm flask. 15 2. Add 0, 5 ml medium from first culture flask which has 19 reached an absorbance of 0. 35 at 660 nm, 3. Incubate as first culture was. TBAB, Difco Laboratories, Detroit, Michigan. J. A. Hoch, Scripps Clinic and Research Fourndation, La JoUa, California. 17 18 19 Antibiotic medium No. 3, Difco Laboratories, Detroit, Michigan. Model G''24 environmental incubator with gyratory shaker, New Brunswick Scientific Co. , New Brunswick, New Jersey. Spectronic 20 spectrophotometer, Bausch and \jomb Inc. , Rochester, New York, 121 C. Microbial Suspension 1. Transfer medium from the second culture flask after it has reached an absorbance of 0. 56-0. 60 to a centrifuge tube. 2. Centrifuge at room temperature a,t 8, 000 x g for 5 min. 3. Pour off supernatant and resuspend organisms with 3 ml of 0. 9% saline for each 10 ml of second culture medium. D. Microbial Assay 1. At time of inoculation of experimental medium, prepare a serial dilution in Spizizen's (13) minimal salts medium of the microbial suspension. 2. E. Plate, in triplicate 0. 1 ml of 10 dilution oqi TBAB plates. Assay of Experimental Medium lt At the conclusion of incubation of experimental rr^edium, prepare a serial dilution in Spizizen's minimal salts medium. 2. Plate in triplicate 0. 1 ml of 10 TBAB plates. -2 and 10 -4 dilution on 1 22 APPENDIX U BACILLUS SUBTILIS GSY 1057 (metB4, hisAl, uvr^l); PARTIAL CONFIRMATION OF PHENOTYPE Streak a fresh TBAB plate with B^ subtiljs from a second culture (Appendix I) which has an absorbance of 0. 56-0. 60, Incubate for 12 hr at 37 . Prepare a TBAB master plate by inoculating 52 stab holes (from a stab repUeator) with separate colonies from the above freshly incubated plate. Incubate for 12 hr at 37 . Transfer with a stab replicatoir inoculum from the fresh 12 hr master plate to triplicate plates with the following media: a) TBAB + 0. 05 \xg/rnl mitomycin C b) Spizizen's minimal salts + 0, 2% glucose c) Spizizen's minimal salts + 0, 2% glucose + 50 [xg/ml histidine d) Spizizen's minimal salts + 0. 2% glucose + 50 jig /ml methionine e) Spizizen's minimal salts + 0. 2% glucose + 50 (j.g/ml histidine and methionine, Incubate the TBAB plates 12-24 hr and the other plates 48 hr. 123 APPENDIX III KREBS RINGER'S SOLUTION ADJUSTED FOR FISH Component Concentrat ion A mourit NaCl 0. 935% 515 KC1 1. 19% 20 2. 19% 5 MgS04- 7H20 3. 97% 5 NaHC03 1. 35% 15 Sodium phosphate bijffer (PH7. 2) 0. 02 M 90 2 4 650 Sodium phosphate buffer (pH 7. 2, 0. 02 M): Na.HPO^ 2 4 1. 47% 4 volumes NaH2PO 1. 43% 1 volume 124 APPENDIX IV NADPH GENERATING SYSTEMS The following highly concentrated generating system gives appreciable activity in trout: For each 3. 2 ml of incubation medium, add 0. 0025 g NADP; 1. 0 nal of glucose-6-phosphate dehydrogenase (G6PdH) plus glucose-6-phosphate (G6P) made up as 0. 5 mg G6PdH (405 units/mg) and 43 mg G6P in 5 ml of 0. 1 M potassium phosphate buffer pH 7. 2; and 1. 0 ml of MgCl (3..03 g/100 ml). Reduced activity is obtained in trout with the foliowit^g system: For each 3. 2 ml of medium, add 0. 1 ml of the following made up in 0, 02 M sodium phosphate buffer pH 7. 2: NADP (0, 01925 g/ml), G6P (0. 00414 g/ml) and G6PdH (0. 000148 g/ml). APPENDIX V 12 5 QUANTITATION OF AFLATOXINS B., B0, R F, R R and Q, 1 2 o o 1 The following data are used in calculating the concentration of the aflatoxins: Toxicant M. W. (nm) (EtOH (3r MLtOH) Reference B 312 360 265 223 21, 800 12, 400 22, 100 248 B2 314 362 265 222 24, 000 12, 100 18, 600 248 R F o 314 331 261 255 13, 700 10, 000 8, 7 90 62 R R o 314 332 261 255 25, 200 19, 000 17, 000 62 328 366 267 223 17, 500 11, 450 19, 030 183 Q1 20 After removing all other solvents, add the selected alcohol to give an approximate concentration of 8-10 |j,g/ml of the toxicant. Measure the absorbance at the applicable wavelength with a suitable recording spectrometer, BeckmanDK-l or PB-GT.21 20 M. S, Masri, WesternRegional Research Laboratory^ Agricultural Research Service, U;>S. Department of Agriculture, Berkeley •keley, California. 21 Beckman Instruments, Inc. , Fullerton, California, 126 The concentration is calculated from / , |jLg/ml =; (A) (M. W. ) (1000) (CF) ! l i ' ' "' ■" ' '' - —"-*■ where A is the absorbance; M. W, is th^ molecular weight of the toxicant; CF is the correction factor for the spectrometer 22 € is the molar absorptivity for the absorbance at the designated wavelength and solvent. As a criteria of purity for B , compare the ra,tios of the absorbances to 220/265 = 1. 77 and 362/265 p 1, 76. 22 Calculated by the method of Rodricks and Stoloff (247). ; 127 APPENDIX VI THIN LAYER CHROMATOGRAPHY: PREPARATIVE AND ANALYTICAL, 1. Prepare TLC plates with one of the following absorbents as indicated by the manufacturer: a) MN-silica gel G-HR23 b) MN-silica gel G-HR F 23 £* O TC 2. "«» c) MN-cellulose powder 300-HR d) Kieselguhr G e) Silica gel plastic sheet 23 23 24 Quickly spot or streak under incandescent light. To assess purity of the fluorescent aflatoxins spot 10 JJII, 15 |J.l and 20 |j.l in duplicate of 200 |j.g/ml of the compounds of interest and appropriate standards. 3. Develop plates in solvent of choice in an unlined chromatography tank protected from light and sudden temperature changes. 23 24 a) Benzene:acetone:ethyl acetate (50:6:12) b) Chloroform:acetone;Lsopropanol (87:10:3) c) Chloroform:acetone:water: isopropanol (88:12:1.5:1.0) d) Chloroformiacetone (9:1) Brinkman Instruments, Inc. , Westbury, New York. , , Chromagram 6060, Eastman Kodak Co. , Rochester, New York, 128 4. e) l-butanol:water:acetic acid (25:15:7) f) Chloroform:methanol (9:1) Locate fluorescent or absorbing compounds with a 365 nm and 254 nm light source. 5. ' To determine amount of label, scrape the spots and count in toluene gel (Appendix XII) or scan with radiochromatogram scanner. 6. Locate I_-staining compounds by developing plates a few min in a TLC chamber saturated with I_ vapor. 7. To identify compounds containing a coumarin moiety (169) spray developed plate with 2 N NaOH, immediately heat at 100 for 5 min and spray with 0.4% £-nitrobenzenediazonium tetrafluoroborate. 27 With a coumarin and NaOH treatment, a deep red color is seen. Without NaOH treatment, a very faint yellow color is observed. 25 26 27 Chromato-vue cabinet, U. V. Model C-5, Ultraviolet Products, Inc. , San Gabriel, California. Blak-Ray UVL-22, Ultraviolet Products, Inc. , San Gabriel, California. Eastman Kodak Co. , Rochester, New York. 129 APPENDIX VII PURIFICATION OF AFLATOXICOL DIASTEREOMERS R F and R R BY COLUMN CHROMATOGRAPHY o o 1. Deactivate aluminum oxide G type E 28 to an activity of Brockmann III by addition of 3% water. Seal and shakje in a round bottom flask with much free heads pace for 1 hr on a wrist action shaker. Equilibrate overnight. Prepare fresh alumina weekly. 2. Slurry 35 g of alumina with approximately 200 ml hexane and pour into a column with dimensions close to 4 x 30 c;m, 3. Add a 2. 5-3. 0 mm layer of fine glass beads to the top of the alumina. 4. Wash the support with an additional 100 ml of hexane. 5. Apply the sample in 1 ml of chloroform. Rinse the sample container with 0. 5 ml of chloroform several times and apply the rinses. 6. Elute the column with 60 ml of hexane with 0. 5 psig N_. 7. Continue to elute with benzene:acetone:ethyl a,cetat^ (50:6:12) i^ntil the R F band has been removed. 8. Elute the R R with benzene:acetone:ethyl acetate (300:40:75), (100:72:48) and (100:72:72). Continue the last system until R R is removed, o 28 Brinkman Instruments, Inc. , Westbury, New York. 130 9. Rechromatograph R F and R R until they are pure by TLC and UV spectroscopy. 131 APPENDIX VIII MASS SPECTRAL ANALYSIS OF AFLATOXICOL DIASTEJREOMERS, R F AND R R o o 1. Prepare sample concentration of approximately 1 (xg /(j.1 acetone or other suitable solvent. 2. Load solid sample cup with approximately 0. 8 ng of sample. 3. Mass spectra are obtained with a Finnigan 1015C GC/MS using the following operating conditions: Solid probe temperature: 70 Ion source temperature: Vacuum: 10 -7 Torr lonization voltage: Mass range: Scan speed: 4. 100 70 ev 12-380 1 sec The significant peaks and suggested assignments are: m/e Assignment 314 Molecular ion 313 Loss of H 296 Loss of HO 268 Loss of HO + CO 267 Loss of H20 + CHO 132 APPENDIX IX NEOTETRAZOLIUM REDUCTASE ASSAY To achieve greater activity with trout use the more concentrated generating system in Appendix IV but replace the MgCl- solution with Li the following neotetrazolium (NT)-MgCl- solution. Dissolve 30 mg NT in a small amount of chloroforrruacetone (1:1). Add 45 ml water. Boil until odor of chloroform is not detected. Add 1. 525 g MgCl and water until 50 ml total volume is reached. For the assay, add 0. 0025 g NADP, 1 ml G6PdH and G6P solution and 1 ml of NT-MgCl_ to a 15 ml conical centrifuge tube. per the tube and incubate for 5 min at 37 . centrifuge tubes in ice, NaCl. min. Transfer 0. 2 ml to Start reaction every 30 sec by adding 0. 1 ml of the desired trout centrifugation fraction. min. Stop- Incubate at 37 for 10 Stop reaction with 3 ml of acetone and add a few crystals of Immediately shake the tube vigorously and centrifuge for 5 Read the absorbance in the UV of the supernatant against a blank at 555 nm. . . Activity = A absorbance x 1000 ——-^-'—-: mg protein x min References: Lester and Smith (163), Maze! (186) and Williams and Kamin (2 92). 133 APPENDIX X ALDRIN EPOXIDATION ASSAY To assay the conversion of aldrin to dieldrin by the 20, 000 x g supernatant, incubate 4. 8 ml of the supernatant, 0. 1 ml DMSO and 250 nmoles aldrin in 0. 1 ml of methyl cellosolve. For inhibition studies with piperonyl butoxide (PB), add the desired concentration in DMSO or in acetone (followed by evaporation) and assay directly or after incubation for 5 min before addition of the aldrin. To measure activity with the top layer microsomal pellet, add 1. 6 ml of the resuspended pellet and the following a.mounts of the more actiye generating system of Appendix IV: and G6P and 1. 6 ml MgCl . 0. 004 g NADP, 1. 6 ml of G6PdH Add aldrin and PB as above. either preparation for 30 min at 25 Incubate on a shaking water bath. ml hexane and shake an additional hr. Add 10 Determine the percent con- version of aldrin to dieldrin by gas chromatographic analysis according to the method of Chan (52). 134 APPENDIX XI DIETS Diet 1 ,(%) Diet 2 (%) Diet 3 (%) 49. 5 38.0 -- Casein -- -- 49. 5 Gelatin 8. 7 9. 0 8. 7 15. 6 -- 15. 6 Dextrose -- 7. 0 „- • b Mineral, mix 2. 5 3. 0 4. 0 CMCC 1. 0 2.0 1. 0 Alpha cellulose 9. 7 18. 0 8. 2 Choline chloride (70%) 1. 0 1. 0 1. 0 Vitamin mix 2. 0 2. 0 2.0 10. 0 20. 0 10. 0 Ingredient Fish protein concentrate (FPC) Dextrin a Herring oil Cerulose, CPC International, Inc. , Englewood Cliffs, New Jersey. Calcium carbonate (CaCC^) (2. 100%); calcium phosphate (CaHPCV 2H2O (73. 500%); potassium phosphate (K2HP04) (8. 100%); potassium sulfate (K2S04) (6.800%); sodium chloride (NaCl) (3. 060%); sodium phosphate (Na2HP04- 6H20) (2. 140%); magnesium oxide (MgO) (2.500%); ferric citrate (FeC6H507-3H20) (0. 558%); manganese carbonate (MnCC^) (0.418%); cupric carbonate (2CuC03Cu(OH)2) (0.034%); zinc carbonate (ZnCC^) (0.081%); potassium iodide (KI) (0.001%); sodium fluoride (NaF) (0.002%); cobalt chloride (C0CI2) (0. 020%); and citric acid (0. 686%). Modified from Bernhart and Tomarelli (29) by addition of the NaF and CoC^. Carboxymethyl cellulose, Hercules Powder Co. , San Francisco, California. rj Thiamine hydrochloride (0. 3200%); riboflavin (0. 7200%); niacinamide (2.5600%); biotin (0.0080%); Ca-pantothenate (D) (1.4400%); pyridoxine hydrochloride (0. 2400%); folic acid (0. 0960%); menadione (0.0800%); B12 (cobalamine-3, 000 fig/g) (0.2667%); i-inositol (meso) (12. 5000%); ascorbic acid (6. 0000%); para-amino-benzoic acid (2.0000%); vitamin D2 (500,000 usp/g) (0.0400%); vitamin A (250,000 lU/g) (0. 5000%); DL alpha tocopherol acetate (2. 5000%); and celite (70. 7293%). APPENDIX XII LIQUID SCINTILLATION COUNTING FLUOR SOLUTIONS 14_ b C counting Efficiency0 Background (%) (cpm) Fluor solution components3. Solution volume (ml) Toluene 1 liter toluene 4 g PPO 40 mg POPOP 15 0-1 Organic 82 26 Toluene gel 1 4 40 40 15 0-1 TLC scrapings 82 26 Name liter toluene g PPO mg POPOP g Cab-O-Sil Sample Volume (ml) Type PPO: 2, 5-diphenyloxazole (Scintillation grade); POPOP: (1, 4-bis-2-(5-phenyloxazolyl)-benzene) (Scintillation grade); toluene (reagent grade); Cab-O-Sil: Godfrey L. Cabot, Inc. , Boston, Massachusetts. Nuclear-Chicago Liquid Scintillation Spectrometer, Nuclear-Chicago, Des Plaines, Illinois. 'Efficiency determined by optimal settings of unquenched sample. standard used to determine efficiency of samples. Reference: Schoenhard (260, p. 85) Channels ratio and internal