Roger A. Coulombe, Jr.

Chemoprevention of Aflatoxicosis in Poultry by Dietary Butylated Hydroxytoluene

Roger A. Coulombe Jr. et al. — Wed, 30 Jun 2010 13:44:45 PDT

Turkeys are among the most sensitive species to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). The programme reported explored whether dietary antioxidants, which have been shown to exert strong chemopreventive properties against AFB1 bioactivity in mammalian models, are likewise protective in turkeys. The feed of 10-day-old male white turkeys were supplemented with BHT (4000 ppm) for the 20 days of the experiment. At the 10th day of pretreatment, AFB1 (1 ppm) was added to some of the diets for another 10 days to give the following groups: control (no treatment), AFB1-only, AFB1 + BHT, and BHT only. Birds in the AFB1-only group had a lower weight gain, a condition which had returned to near control in groups fed diets containing AFB1 + BHT. Activity of hepatic a microsomal cytochrome P450 (CYP) 1A homologue as well as conversion of AFB1 to the putative toxic metabolite, the exo-AFB1-8,9-epoxide (AFBO) were significantly lower in the BHT group compared to control. Conversely, dietary BHT significantly increased activities of the phase II enzymes glutathione S-transferase, as well as quinone oxidoreductase (QOR). However, there was no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. Dietary AFB1 caused diffuse hepatocellular necrosis and biliary hyperplasia, the severity of which was significantly lessened in the AFB1 + BHT treated group. Slight hepatocellular hydropic degeneration was observed in the BHT-only group, but not in the AFB1 + BHT groups. This condition associated with BHT treatment was found in a separate study to be reversible and without any long-term adverse effects. Despite induction of phase II enzymes in turkey liver, our data indicates that the strong chemopreventive ability of BHT appears to occur via primarily inhibition of AFB1 activation, while having no measurable effect toward specific AFB1 detoxification by GST. Thus, BHT and possibly related phenolic antioxidants may prove to be a viable feed additive for the reduction of aflatoxicosis in poultry.

Comparative DNA Cross-Linking by Activated Pyrrolizidine Alkaloids

H. Y. Kim et al. — Wed, 30 Jun 2010 13:44:45 PDT

The toxicity and bioactivity of pyrrolizidine alkaloids (PAs), common constituents of hundreds of plant species, and in herbal remedies and folk medicines prepared thereof, are probably due to their ability to form DNA cross-linking. We investigated DNA cross-linking activity by chemically-activated PAs from four different structural classes in Madin–Darby bovine kidney (MDBK) cells and in pBR322 DNA. In cell culture, α,β-unsaturated macrocyclic diester pyrroles dehydrosenecionine (DHSN), dehydroriddelliine (DHRD) and the saturated macrocyclic diester pyrrole dehydromonocrotaline (DHMO) were significantly more potent cross-linkers than the simple necine base (retronecine) and an N-oxide (indicine N-oxide; INO) as determined by alkaline elution. The proportion of total DNA cross-links that were proteinase K-resistant (DNA–DNA cross-links) induced by the various pyrroles ranged from 0.08 (DHRN) to 0.67 (DHSN). Those pyrroles that were potent cross-linkers of cellular DNA also cross-linked, in a dose-dependent manner, Bam H1-digested pBR322 DNA as assessed by a gel retardation assay. The possible functional relevance of pyrrole–DNA cross-links was determined by their ability to interrupt PCR amplification of a 1.129 kb segment of pBR322. Dehydrosenecionine completely inhibited amplification, while DHMO was of intermediate potency, while DHRN and INO had no effect. Taken together, these studies suggest that structural features, most notably the presence of a macrocyclic diester, confer potent cross-link activity to PAs. In any event, DNA–DNA cross-linking is probably biologically relevant as indicated by their interference with DNA replication.

Effects of Dietary Butylated Hydroxytoluene on Aflatoxin B1-Relevant Metabolic Enzymes in Turkeys

P. J. Klein et al. — Wed, 30 Jun 2010 13:44:44 PDT

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B1 (AFB1) is due to a combination of efficient AFB1 activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB1-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB1 to the putative toxic metabolite, the exo-AFB1-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB1-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB1 detoxification by GST.

Antioxidants Protect Turkeys Against Toxicity of Aflatoxin

Roger A. Coulombe Jr. — Wed, 30 Jun 2010 13:44:43 PDT

Toxins stemming from mold in feed grains are unavoidable in poultry production. "Mycotoxins" such as aflatoxin B (AFB1) are among the most potent liver-damaging toxins known. AFB1 is also a probable human carcinogen. Poultry are the most sensitive of all farm animals to the toxic effects of even small amounts of AFB1. Although poultry don’t generally live long enough to develop cancers, AFB1-related diseases adversely affect their health. This aflatoxin can cause slowing of growth and decreased resistance to microbial pathogens that make poultry ill. Because these toxins are so pervasive in feed grains, eliminating them has proved either impractical or prohibitively expensive. Mycotoxins cost the poultry industry more than $100 million annually in productivity losses and reduced product quality. These losses have been absorbed into the cost of production. Aflatoxins such as AFB1–used in this study –are also known as "pro-toxins." That is, they are not toxic in their original state, but they become so only after being eaten and then reacting with liver enzymes. But there are also protective enzymes in the liver. In some animals that are AFB1-resistant, the active form of AFB1 is efficiently detoxified by a group of enzymes called glutathione S-transferases (GSTs).

Pyrrolizidine Alkaloids Plants, Metabolism and Toxicity

B. L. Stegelmeier et al. — Wed, 30 Jun 2010 13:44:43 PDT

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.

Butylated Hydroxytoluene Chemoprevention of Aflatoxicosis - Effects on Aflatoxin B1 Bioavailability, Hepatic DNA Adduct Formation, and Biliary Excretion

J. A. Guarisco et al. — Wed, 30 Jun 2010 13:44:42 PDT

The extreme sensitivity of turkeys to aflatoxin B1 (AFB1) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB1 toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB1 bioactivation. To test whether dietary BHT alters hepatic AFB1–DNA adduct formation, excretion, and bioavailability of AFB1 in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [3H]-AFB1 (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB1–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M1 (AFM1) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB1, aflatoxin Q1 or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB1 bioavailability in addition to inhibition of bioactivation.

Comparative Aflatoxin B1 Activation and Cytotoxicity in Human Bronchial Cells Expressing Cytochromes P450 1A2 and 3A4

Terry R. Van Vleet et al. — Wed, 30 Jun 2010 13:44:42 PDT

Some epidemiological evidence suggests a link between the inhalation of aflatoxin B1 (AFB1)-contaminated grain dusts and increased lung cancer risk. However, the mechanisms of AFB1 activation and action in human lung are not well understood. We compared AFB1 action in SV40 immortalized human bronchial epithelial cells (BEAS-2B) with two transfected cell lines that stably express human cytochromes P450 (CYPs) 1A2 (B-CMV1A2) and 3A4 (B3A4), the principal CYPs thought to activate this mycotoxin in human liver. All three cell types retained catalytically active glutathione S-transferase, the key phase II enzyme that detoxifies metabolically activated AFB1. B-CMV1A2 and B3A4 cells expressed methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase activities, respectively, and were 3000- and 70-fold more susceptible, respectively, to the cytotoxic effects of AFB1 than the control cell line (BEAS-2B). When cultured with a range of low, environmentally relevant AFB1 concentrations (0.02–1.5 µM), control cells formed barely detectable AFB1-DNA adducts, whereas B-CMV1A2 cells formed significantly more adducts than B3A4 cells. In B-CMV1A2 cells, formation of AFB1-DNA adducts was inhibited by the CYP 1A2 inhibitor 7,8-benzoflavone, whereas formation of AFB1-DNA adducts in B3A4 cells was inhibited by the CYP 3A4 inhibitor 17{alpha}-ethynylestradiol. Competitive reverse transcription-PCR analysis showed that only the CYP-transfected cell lines expressed CYP mRNA. When adjusted for CYP mRNA expression, B-CMV1A2 cells were more efficient in the formation of cytotoxic and DNA-alkylating species at low AFB1 concentrations, whereas B3A4 cells were more efficient at high concentrations. Our results affirm the hypothesis that, as in human liver microsomes, CYP 1A2 in human lung cells appears to have a more important role than CYP 3A4 in the bioactivation of low AFB1 concentrations associated with many human exposures. Therefore, it is possible that under conditions in which appropriate CYPs are expressed in lung, inhalation of AFB1 may result in increased risk of lung cancer in exposed persons.

Effects of Repeated Doses of Aspartame on Serotonin and its Metabolite in Various Regions of the Mouse Brain

R. P. Sharma et al. — Wed, 30 Jun 2010 13:44:41 PDT

Following a finding that single doses (approximating to average intakes and to potential ‘over-use’) of aspartame administered orally to mice caused significant increases in norepinephrine and dopamine concentrations in various brain regions, the effect of repeated exposure to aspartame was studied. Male CD-1 mice were given a daily oral dose of 0, 13, 133 or 650 mg/kg for 30 days and 1 day after the last dose the animals were decapitated and their brain regions were quickly isolated. Analyses of the different regions for catecholamine and indoleamine neurotransmitters and their major metabolites indicated that the increases in adrenergic chemicals observed shortly after a single exposure were not apparent after repeated dosing. In contrast, concentrations of serotonin and its metabolite, 5-hydroxyindoleacetic acid, were decreased in several regions. An increased supply of phenylalanine may be responsible for a decrease in tryptophan uptake by the brain tissue or for a depression in tryptophan conversion to serotonin.

Oxidation of NADH by Vanadium in the Presence of Thiols

R. J. Keller et al. — Wed, 30 Jun 2010 13:44:40 PDT

The nonenzymatic oxidation of NADH was studied spectrophotometrically in the presence of two vanadium compounds, sodium orthovanadate and vanadyl sulfate. At physiological pH 7.4, in 25 mImage sodium phosphate buffer, addition of the synthetic thiol, dithioerythritol (DTE) results in a marked increase of NADH oxidation in the presence of sodium orthovanadate, but not in the presence of vanadyl sulfate. Other reductants, such as dithiothreitol and cysteine, can also increase NADH oxidation, whereas glutathione and ascorbate cannot. In all reactions, superoxide dismutase and catalase completely inhibit the vanadium-stimulated oxidation of NADH. Inhibition occurs in a concentration-dependent manner, and the boiled enzymes do not inhibit the thiol reaction. The hydroxyl radical scavenger, thiourea, inhibits the reaction, whereas urea cannot. ESR studies show that the ability of the thiol to reduce vanadate can be correlated with the degree of NADH oxidation. Using spin trapping techniques, hydroxyl radicals are detected during the course of the reaction. Addition of hydrogen peroxide to vanadyl in the presence of DTE greatly increases NADH oxidation; however, no NADH oxidation occurs when hydrogen peroxide is added to vanadyl and ascorbic acid. These results provide a partial explanation for the ability of vanadium compounds to both decrease cellular reducing equivalents and promote lipid peroxidation.

Importance of Hydroxyl Radical in the Vanadium-Stimulated Oxidation of NADH

R. J. Keller et al. — Wed, 30 Jun 2010 13:44:40 PDT

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO—OH adduct formed by vanadate plus superoxide. these results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.

Urban Particulate Matter Activates Calpain and Triggers the Unfolded Protein Response

T. L. Watterson et al. — Wed, 30 Jun 2010 13:44:39 PDT

Structural Influences on Pyrrolizidine Alkaloid Induced Cytopathology

H. Y. Kim et al. — Wed, 30 Jun 2010 13:44:38 PDT

Pyrrolizidine alkaloids (PAs), which are common constituents of hundreds of plant species around the world, have been reported to have cytotoxic, carcinogenic, antineoplastic, and genotoxic activity in vivo and in vitro. The exact mechanism of these biological toxicities is not yet clear. The ability of eight PA congeners to inhibit mitosis and induce megalocyte formation in cultured bovine kidney epithelial cells was studied to examine possible structural influences on these endpoints. Representatives of the three PA structural groups, the macrocycles (seneciphylline, senecionine, riddelliine, retrorsine, monocrotaline), open diesters (heliosupine, latifoline), and a necine base (retronecine), were cocultured for 2 hr with a NADPH-generating system and rat liver S9. Macrocylic PAs with αβ-unsaturation (seneciphylline, senecionine, riddelliine, retrorsine) showed a dose-dependent inhibition of colony formation at 50, 100, and 300 μM and induction of megalocytosis at 500 μM. Colony growth resumed 3 weeks after removal of PAs at 50 and 100 μM, and normal cellular morphology returned 5 or 6 weeks after removal of PAs at 500 μM. The saturated macrocyclic (monocrotaline) and open diesters (heliosupine, latifoline), elicited only a slight inhibition of colony formation and had no effect on cellular morphology at 500 μM. The necine base (retronecine) had no effect on either colony formation or cell morphology. Pyrrolic PAs (dehydrosenecionine, dehydromonocrotaline, dehydroretronecine) were more active in inhibition of colony formation than their parent compounds and were potent inducers of abnormal cellular morphology at 500 μM. An N-oxide metabolite, indicine N-oxide, was completely inactive. The results support previous studies showing that there are structural influences on PA-induced cytopathological effects.

Mechanisms of Butylated Hydroxytoluene Chemoprevention of Aflatoxicosis - Inhibition of Aflatoxin B1 Metabolism

John A. Guarisco et al. — Wed, 30 Jun 2010 13:44:37 PDT

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B1 (AFB1) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB1 metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB1 to the reactive intermediate AFB1-8,-9-epoxide (AFBO), exhibiting Michaelis–Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB1 in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB1 epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.

Metabolism and Cytotoxicity of Aflatoxin B1 in Cytochrome P-450-Expressing Human Lung Cells

Terry R. Van Vleet et al. — Wed, 30 Jun 2010 13:44:36 PDT

The mycotoxin aflatoxin B 1 (AFB 1 ) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB 1 has been detected in dusts generated in the processing and transportation of AFB 1 -contaminated products. Inhalation of grain dusts contaminated with AFB 1 may be a risk factor in human lung cancer. Aflatoxin B 1 requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB 1 activation in human lung is not well understood. To investigate whether these CYPs can activate AFB 1 at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 n M AFB 1 produced the AFB 1 -glutathione conjugate (AFB 1 -GSH) and aflatoxin M 1 (AFM 1 ), while B3A4 cells produced only aflatoxin Q 1 (AFQ 1 ) at 0.15 µ M AFB 1 . Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 m M AFB 1 . Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB 1 to AFB 1 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S -transferase (GST)-mediated trapping of enzymatically generated AFB 1 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB 1 compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB 1 , followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB 1 primarily at higher AFB 1 concentrations, was also responsible for significant AFB 1 toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB 1 , even at environmentally relevant concentrations.

Genes Associated with Aflatoxin B1 Sensitivity

Roger A. Coulombe Jr. et al. — Wed, 30 Jun 2010 13:44:33 PDT

Metabolism of Aflatoxin B1 in the Upper Airways of the Rabbit: The Role of the Non-Ciliated Tracheal Epithelial Cell

Roger A. Coulombe Jr. et al. — Wed, 30 Jun 2010 13:44:33 PDT

Short-term tracheal explant cultures from the rabbit were used to study the metabolism of the carcinogen aflatoxin B1 (AFB1) and to determine the cell types that are susceptible to damage by AFB1 and their relative contents of monooxygenase enzymes. Tracheas were cultured in serum-free medium for 0.5–24 h with 0.7 µM [14C]AFB1, and metabolism was measured by determining the level of binding of the carcinogen to DNA and by the release of metabolites into the medium. The binding of aflatoxin B1 was time dependent and appeared to peak at 12 h in culture. In addition, the metabolites aflatoxicol, aflatoxin M1, and aflatoxin Q1 were produced by the explants. Ultrastructural evaluation of cultured tracheas showed degenerative changes exclusively in nonciliated secretory cells after 4 h in culture. Extensive nonciliated secretory cell necrosis was evident by 12 h. Ciliated cells did not show degenerative changes until 12 h and appeared much more viable after 24-h exposure to AFB1 relative to the nonciliated cells. Tracheal sections stained to demonstrate rabbit lung cytochrome P-450, Forms 2 and 5, and cytochrome P-450 reduced nicotinamide adenine dinucleotide phosphate reductase by an immunoperoxidase technique showed intense staining selectively within nonciliated cells. In total, the data revealed that: (a) rabbit tracheal explants are able to metabolize aflatoxin B1; (b) the nonciliated secretory cell population in this tissue is the target cell for cytotoxicity of this carcinogen; and (c) as is the case in the more distal airways, the nonciliated epithelial cells appear to have a high content of components of the pulmonary cytochrome P-450 monooxygenase system, which may be an important factor in the susceptibility of these cells and this region of the airways to suspected airborne carcinogens.

Aflatoxin B1 Alters the Expression of P53 in Cytochrome P450-Expressing Human Lung Cells

Terry R. Van Vleet et al. — Wed, 30 Jun 2010 13:44:32 PDT

Aflatoxin B1 (AFB1) is a potent dietary hepatocarcinogen in animals and probably in humans. Mutations (and altered expression) of the tumor suppresser gene p53 have been observed in liver tumors from patients exposed to high dietary AFB1. Inhalation of AFB1-laden grain dusts has been associated with an increased incidence of lung cancer in humans as well. We examined the effects of low concentrations of AFB1 on the expression of p53 and MDM2 in human bronchial epithelial cells (BEAS-2B) transfected with cDNA for either cytochrome P450 (CYP) 1A2 (B-CMV1A2) or CYP 3A4 (B3A4), two isozymes that are responsible for AFB1 activation in human liver and possibly the lung. Untreated B-CMV1A2 and B3A4 cells constitutively expressed p53. Exposure to a range (0.015–15 µM for 30 min) of AFB1 concentrations caused a concentration-dependent decline in p53 expression in B-CMV1A2 cells, and to a lesser extent, in B3A4 cells. The AFB1-mediated decrease in p53 continued for at least 12 h after 30-min exposures to 1.5 µM AFB1. Mirroring the decrease in p53 expression was a concentration-dependent increase in the expression of the 76-kDa MDM2 isoform in B-CMV1A2 and B-3A4 cells. Interestingly, AFB1 did not induce DNA laddering, an indicator of apoptotic cell death, but proteolytic activation of caspase-3 was detected in AFB1-treated B-CVM1A2 cells. In total, these data show that low, environmentally-relevant concentrations of AFB1 alter the expression of p53 and MDM2 in these human lung cells, and that cells that stably express CYP 1A2 were more susceptible to this effect than nontransfected, or 3A4-expressing cells.

Aflatoxin B1 Activation in Human Lung

Jack D. Kelly et al. — Wed, 30 Jun 2010 13:44:32 PDT

Inhalation exposure to the carcinogen aflatoxin B1(AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1inhalation may cause primary lung cancer, we investigated AFB1activation by human lung microsomes. Microsomes were incubated with [3H]AFB1(124 μImage ), and activation to the AFB1-8,9-epoxide was measured as the AFB1–glutathione (AFB1–GSH) conjugate by HPLC. The formation of AFB1–GSH was in the range of 0.05–0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01–1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1to form theexo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung.In situAFB1activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Butylated Hydroxytoluene Chemoprotection: Response to Williams

Roger A. Coulombe Jr. et al. — Wed, 30 Jun 2010 13:44:30 PDT

Letter to the Editor.

Metabolism of Aflatoxin B1 by Normal Human Bronchial Epithelial Cells

Terry R. Van Vleet et al. — Wed, 30 Jun 2010 13:44:30 PDT

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB1 have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB activation in human lung, cytochrome P-450 (CYP)-mediated activa1 tion and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0.15 µM or 1.5 µM AFB1 for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0.15 µM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1(AFQ1). At 1.5 µM AFB1, both aflatoxin M1(AFM1), and AFQ1 were produced. Cells pretreated with 50 µM 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 µ M AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 µM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S -transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate AFB1 inefficiently, but possess CYPs reportedly responsible for metabolism of AFB1. These data support earlier findings showing modest CYP-mediated AFB1 activation in human airways, but indicate that exposure to polycyclic aromatic hydrocarbons (PAHs), such as 3MC, which induce CYP(s) that specifically activate AFB1 may increase the harmful effects of AFB1 exposures in human airways.