Hira L. Gurtoo

Effects of inducers and inhibitors on the metabolism of aflatoxin B, by rat and mouse

Abstract The microsomal metabolism of aflatoxin B1 (AFB1) via various pathways and the induction and inhibition specificities of these pathways were examined in Sprague-Dawley rats, inbred strains of mice, and recombinant inbred lines derived from AKR/J and C57L/J cross. The data suggests that the metabolism of aflatoxin B1 is catalyzed by at least three different enzymes of the microsomal mixed function oxygenase system: one that mediates conversion to aflatoxin M1 (AFM1-hydroxylase) is cytochrome P-448-linked and is associated with the Ah locus at the level of regulatory genetic factors: the other two enzymes which are cytochrome P-450-linked, mediate the metabolism of aflatoxin B1 to aflatoxin Q1 (AFQ1-hydroxylase) and to AFB1-2,3-oxide (metabolic activation). Although AFQ1-hydroxylase and metabolic activation, measured in vitro as the formation of DNA-alkylating metabolite(s), are not clearly distinguishable on the basis of induction and inhibition responses, the two activities can be distinguished on the basis of their kinetic parameters and genetic regulation: (a) the apparent Km of the metabolic activation pathway is almost three orders of magnitude (19.7 × 10−4 M) higher than that of AFQ1-hydroxylase (0.7 × 10−4), [B. D. Roebuck and G. N. Wogan, Cancer Res.37, 1649 (1977)]; and (b) while both activities are induced by phenobarbital (PB) and depressed by 3-methylcholanthrene (3-MC), unlike the metabolic activation, differences in the 3-MC-induced depression of AFQ1-hydroxylase activity were noted in Ah responsive and nonresponsive strains. Kinetic studies revealed that metabolic activation of AFB1 is linear with the time of incubation up to 20 min, with up to 2 mg of microsomal protein in the incubation, and has a pH optimum of 7 to 7.4. While PB pretreatment, relative to control, enhanced the apparent Vmax 3-fold, 3-MC depressed it about 60 per cent; however, the apparent Km values of the three microsomal preparations were of the same order of magnitude (1.3 to 2 × 10−3 M). Under the conditions of the incubation, the minimum number of binding sites in DNA was estimated to correspond to a resultant specific activity of 8–9 nmoles AFB1-metabolite bound/μmole DNA-P.

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Abstract Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. In vitro CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.

Genetic expression of aflatoxin metabolism: effects of 3-methylcholanthrene and β-naphthoflavone on hepatic microsomal metabolism and mutagenic activation of aflatoxins

The effects of pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (beta NF) on the hepatic microsome-mediated mutagenesis of aflatoxin B1 (AFB1) and benzo[a]pyrene, and on the metabolism of aflatoxins B1 and B2, were investigated in inbred mouse strains. The inbred strains of mice studied included Ah nonresponsive strains (DBA/2Ha, AKR/Sn and RF/J), which were also nonresponsive to the induction of the metabolism of AFB1 to AFM1 (AFB1-4-hydroxylase activity), and Ah responsive strains (C57BL/6Ha, ICR/Ha, C3H/St, A/St, Balb/cCr, C57e/Ha and CBA/Pi), which were also responsive to the induction of AFB1-4-hydroxylase activity. The hepatic microsome-mediated enzyme activities studied included: mutagenic activation of AFB1 and benzo[a]pyrene in the Ames Salmonella typhimurium TA-98 system; metabolism of AFB1 and AFB2 to AFM1 and AFM2, respectively; and benzo[a]pyrene metabolism measured as the formation of fluorescent phenolic metabolites, i.e. aryl hydrocarbon hydroxylase (AHH) activity. Time-course and dose-response studies in C57BL/6Ha mice revealed that the metabolism of aflatoxin B1/B2 to aflatoxin M1/M2 (AFB1/B2-4-hydroxylase activity) was induced by both MC and beta NF. In the nonresponsive strains studied, pretreatment with MC or beta NF produced essentially little alteration of AFB1-4-hydroxylase activity or AHH activity or the mutagenic activation of AFB1 and benzo[a]pyrene. On the other hand, AFB1-4-hydroxylase activity in the responsive strains was induced 4- to 10-fold by MC (60 mg/kg) and 2.5- to 7-fold by beta NF (150 mg/kg). Also in the responsive strains, induction of AFB1-4-hydroxylase activity was strongly associated with (a) the depression of the mutagenic activation of AFB1, and (b) with the induction of both AHH and the mutagenic activation of benzo[a]pyrene. In summary, the results described in this report suggest that: (a) induction of AFB1-4-hydroxylase activity by MC (or beta NF) is associated with the depression of AFB1 mutagenesis and with the induction of benzo[a]pyrene mutagenesis; and (b) induction by MC (or beta NF) of AHH activity, AFB1-4-hydroxylase activity and AFB2-4-hydroxylase activity is controlled by either the same or closely linked genetic factors.

High pressure liquid chromatographic separation of multiple forms of cytochrome P-450

The major form of cytochrome P-450 isolated and purified from the hepatic microsomes of phenobarbital pretreated rats by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns was found to be homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. However, this cytochrome P-450 was resolved into three bands by high pressure liquid chromatography on an Anpac ion-exchange column. High pressure liquid chromatography isolated forms had similar molecular weights of 55,000 with lambda max of the CO-reduced difference spectrum at 450 nm and were found to be in the low spin state. The results demonstrate the effectiveness of high pressure liquid chromatography in the resolution of cytochrome P-450s of similar molecular weights but different net charges.

Resolution of multiple forms of cytochrome P-450 by high-performance liquid chromatography

The major forms of cytochrome P-450 in the hepatic microsomes of rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC) were isolated by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns. These preparations exhibited single protein bands corresponding to cytochrome P-450s by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). High-performance liquid chromatography (HPLC) of these preparations on an anion-exchange column yielded three peaks from the PB-induced major cytochrome P-450 and a single peak from the 3MC-induced major cytochrome P-450. That the HPLC-isolated protein peaks were various forms of cytochrome P-450 was confirmed by spectral examination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Examination of their absolute spectra revealed these cytochrome P-450s to be in a low-spin state. The lambda max of the reduced CO-complex spectra and molecular weights were found to be 450 nm and 53,000, respectively, for all the three HPLC-resolved cytochrome P-450s from PB-induced rats; and 448 nm and 56,000, respectively, for the HPLC-isolated cytochrome P-450 from 3MC-induced rats. The results demonstrate the effectiveness of HPLC in the determination of cytochrome P-450 multiplicity and charge heterogeneity.

On the binding of aflatoxin B1 and its metabolites to hepatic microsomes

Abstract The metabolism of aflatoxin B 1 was studied using the cytochrome P450-dependent mixed function oxidase system of rat liver microsomes. An aflatoxin metabolite produced in the presence of microsomes and NADPH and not produced in the presence of SKF-525A seems to become covalently bound to microsomes. The bound metabolite is observed as a spectral peak at 412 nm by means of difference spectroscopy. This metabolite appears to be related to either aflatoxin B 2a or its precursor.

Genetic dependence of hepatic microsome-mediated depression of aflatoxin B1 activation to mutagens in Ames Salmonella typhimurium TA-98 system.

Pretreatment of C57B1/6, an Ah-responsive strain of mice, with 3-methyl cholanthrene or β-naphthoflavone caused a depression of the hepatic microsome-mediated metabolism of alfatoxin B1 to metabolites mutagenic to Salmonellathyphimurium TA-98 strain, while the activity of hepatic mecrosomes from an Ah nonresponsive strain DBA/2 was unaffected by these pretreatments. In contrast to results obtained with aflatoxin B1, 3-methylcholanthrene or β-napthoflavone pretreatment of C57B1/6 enhanced several fold the metabolism of benzo(a)pyrene to mutagenic metabolites in this system, whereas pretreatment of DBA/2, like that seen with aflatoxin B1, did not affect the formation of mutagenic metabolites of benzo(a)pyrene.

Metabolism of a prototype mycotoxin, aflatoxin B1, and its genetic regulation.

Toxins of diverse chemical structures produced by various types of fungi have generally remained unnoticed because of the sporadic nature of mycotoxicoses and uncertainties surrounding identification of mycotoxins. The occurrence of mycotoxins in the environment is dertermined not only by the geographical location but also by methods of cultivation, harvest, storage, preservation and preparation of animal feeds and human foods. Thus, warm and humid conditions that promote fungal growth invite mycotoxin contamination and when large quantities of foodstuffs are involved mycotoxicoses may reach epidemic proportions and evoke public concern. Perhaps best known and earliest recognized example of mycotoxicoses is that of Ergotism caused by toxic alkaloids of Claviceps purpurea (1). Stachybotryotoxicosis in the early 1930s killed thousands of horses in the Ukraine and also affected man (2). Between 1943-1947 toxins produced by some species of Fusarium and Cladosporium on the grain, from which bread was made, led to wide-spread outbreaks of a fatal hemorrhagic disease, alimentary toxic aleukia, in Russia (2). A classical example of a wide-spread outbreak of mycotoxicoses is that of aflatoxins in Brazilian peanut meal which was incriminated in the death of hundreds of thousands of turkey poults and ducklings in England (3). In addition to their toxicity, mycotoxins may also be teratogenic and carcinogenic (2). Aflatoxins, which are both hepatoxic and hepatocarcinogenic (4) are among the most potent mycotoxins. Four principal aflatoxins, AFB~ and AFB 2 having blue fluorescence, and A F G a and A F G 2 having green fluorescence (Fig. 1), are produced by Aspergillus flavus and

Differential sensitivity of ah‐responsive mice to β‐naphthoflavone‐induced metabolism and m utagenesis of benzo[a]pyrene and aflatoxin B1

Effects of the administration to C57BL/6ha (Ah-responsive) mice of a low (10 mg/kg) and a high dose (150 mg/kg) of beta-naphthoflavone (BNF) on the hepatic microsome-mediated mutagenesis and metabolism of benzo[a]pyrene (BP) and aflatoxin B1 (AFB1) were studied. Hepatic microsome-mediated mutagenesis of benzo[a]pyrene was not enhanced by the low dose (10 mg/kg) but at the high dose (150 mg/kg) the mutagenic activation was enhanced several fold relative to control (corn oil-treated). Mutagenic activity of aflatoxin B1 was however depressed by both the low and the high doses of beta NF. These results are consistent with the effects of beta NF administration on hepatic microsome-mediated metabolism of BP to its phenolic products and on the metabolism of aflatoxin B1 to aflatoxin M1 catalyzed by aflatoxin B1-4-hydroxylase. Relative to control, pretreatment of the mice with 10 mg/kg beta NF did not induce aryl hydrocarbon hydroxylase activity (a measure of BP metabolism), however, the same pretreatment induced the metabolism of AFB1 to AFM1 by 2.7 to 4.7-fold. Microsomal preparations from 150 mg/kg beta NF-pretreated mice showed a 3-fold induction of aryl hydrocarbon hydroxylase activity and a 6.8-fold induction of AFB1-4-hydroxylase activity. These results suggest that two different enzyme systems are involved in the metabolism of BP and the metabolism of AFB1 to AFM1.

The effect of inducers of mixed-function oxidases on hepatic microsome-mediated aflatoxin B1 transformation in C3H10T12 cells

The potent hepatotoxin and hepatocarcinogen aflatoxin B1 (AFB1) is metabolized by different forms of cytochrome P450 associated with the hepatic mixed-function oxidase system. C3H/10T1/2(10T1/2) cells, which have limited inherent capacity to metabolize AFB1, were treated with AFB1 in the presence of hepatic microsomes isolated from chemically treated rats to investigate the effects of the induction of specific cytochromes P450 on AFB1-mediated toxicity and transformation. Relative to uninduced microsomes, phenobarbital (PB) treatment induced AFB1-DNA binding (essentially representing the formation of AFB1-8,9-oxide bound to DNA) 3.2-fold, while pretreatment with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (beta-NF) preferentially induced aflatoxin M1 (AFM1) formation from 2- to 5-fold. 10T1/2 cells were exposed to a multiple treatment regimen with 4 microM AFB1 and hepatic microsomes from uninduced, PB-, Aroclor 1254-, 3-MC-, or beta-NF-treated rats; respective cumulative toxicities of approximately 90, 95, 70, 60, and 40% control (no microsomes) values resulted. An enhanced AFB1 transformation response correlated with the increasing toxicities observed for the different treatments, with uninduced or PB-induced microsomes yielding approximately four foci/dish, while treatments with Aroclor 1254-, 3-MC-, or beta-NF-induced microsomes resulted in only one to two foci/dish. These results demonstrate that AFB1 is a complete carcinogen in the 10T1/2 system when repetitive incubations are used in conjunction with an appropriate hepatic microsomal activation system. These data also correlate the induction of AFB1-4-hydroxylase with a decrease in AFB1-mediated toxicity and transformation of 10T1/2 cells, and support the hypothesis that the Phase I metabolic conversion of AFB1 to AFM1 in the liver represents an effective detoxification pathway per se.