Metabolism, DNA binding, and cytotoxicity of aflatoxin B1 in tracheal explants from Syrian hamster

Toxicology, 32 (1984) 117--130 Elsevier Scientific Publishers Ireland Ltd.

METABOLISM, DNA BINDING, AND CYTOTOXICITY OF AFLATOXIN B1 IN TRACHEAL EXPLANTS FROM SYRIAN HAMSTER

ROGER A. COULOMBE, JR.*, DENNIS W. WILSON and DENNIS P.H. HSIEH**

California Primate Research Center, University of California, Davis, CA 95616 (U.S.A.) (Received December 19th, 1983) (Accepted February 29th, 1984)

SUMMARY

Metabolism, DNA binding and cytopathological effects of aflatoxin B1 (AFB~) were studied in isolated tracheal explants from Syrian golden hamsters. Explants were exposed to 0.1, 0.5 and 1.0 uM [14C]AFB~ in Dulbeccos's modified Eagle medium for 24 h, then analyzed for AFB~-DNA binding and AFBI metabolism. Binding (pmol AFB~/mg DNA) was doserelated {16.3 + 1.9 to 180.8 + 16.1) and analysis of the culture medium revealed the metabolic conversion products aflatoxicol (AFL) and aflatoxin QI (AFQ~). Ultrastructural analysis of sections of tracheal epithelium revealed degenerative changes primarily in the non-ciliated epithelial cells. Autoradiographic analysis of the same treated explants, however, showed no discernible distribution of label with respect to either cell type or cell location, with the exception of increased grain densities overlying vacuoles containing dark droplets. In addition, $9 prepared from hamster trachea was shown to activate AFBI to mutagens detectable by Salmonella typhimurium TA 98, but was approximately 70 times less active on a per mg protein basis than was $9 prepared from hamster liver. These results demonstrate the metabolic capabilities of tracheal epithelial cells in the activation of AFB~, thus indicating that AFB~ present in respiratory particles may be activated by pulmonary mixed-function oxidases, posing a hazard to those exposed.

Key words: Aflatoxin B1; Covalent binding; Metabolic activation; Tracheal explant culture; Ultrastructural changes *Present address: Department of. Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, U.S.A. **To whom reprint requests should be sent. Abbreviations: AFBI, aflatoxin BI; AFL, aflatoxicol; AFMI, aflatoxin MI; AFQ~, ariatoxin Q l; IAC, isoamylalcohol-chloroform; MFO, mixed function oxidase; SER, smooth endoplasmic reticulum. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

117

INTRODUC~ON

Aflatoxin B1 (AFB1), a mycotoxin produced by strains of Aspergillus flavus and A. parasiticus, is a potent carcinogen in a wide number of species [1] producing tumors in the liver as well as other organs [2]. AFB~ is metabolically converted to a variety of stable metabolites including aflatoxicol (AFL), aflatoxin M1 (AFM,) and aflatoxin Q1 (AFQ~), in vivo or in vitro when incubated with a NADPH-generating system and cytochrome P-450containing tissue preparations [ 3 ]. Metabolic activation by the cytochrome P-450-containing mixed-function oxidases (MFO) is a requisite step in the formation of the putative active intermediate, the AFBr8,9-epoxide [4], reportedly responsible for several biological effects including carcinogenicity [5], DNA binding [6], cytotoxicity [ 7], and bacterial mutagenicity [8]. Although humans and animals are exposed to AFB, primarily via the diet, considerable evidence underlies the possibility that AFB1, present in respirable particles, may be activated by pulmonary MFOs and hence pose a potential carcinogenic hazard by this route. Sorenson and workers [9] found AFB~ in levels as high as 1814 ppb in respirable, airborne grain dusts collected from grain terminals in the midwestern United States. Dvorackova [10] reported 2 cases of human alveolar cell carcinoma presumably associated with inhalation of AFB,-contaminated peanut meal; extracts from samples of lung tissue taken at autopsy were shown to contain AFB~. Moreover, grain dock workers have been observed to exhibit an elevated frequency of bronchial carcinoma relative to the normal population [11] although the underlying etiology has not been rigorously established. The objectives of this study were to evaluate metabolism, DNA binding, and cytotoxicity of AFB~ in cultured intact tracheal epithelium. Explant cultures from hamsters were used as a model since previous findings from our laboratory [12] indicated that relative to other common laboratory animals, the upper airways from this species possess a greater proportion of non-ciliated epithelial cells containing abundant smooth endoplasmic reticulum (SER). Similar cells appear to be a major site of xenobiotic metabolizing activity in the distal pulmonary airways [ 13]. M A T E R I A L S AND METHODS

Animals Male Syrian Golden hamsters weighing 100--130 g were purchased from Simonsen laboratories (Gilroy, CA). They were maintained on Purina laboratory chow No. 5001 and water ad libitum. Chemicals Ring labeled [14C]AFB1 was prepared from cultures of Aspergillus parasiticus ATCC 15517 supplemented with [ 14C]acetate (New England Nuclear, Boston, MA) as described by Hsieh and Mateles [14]. No UV-absorbing or 118

fluorescent impurities were evident as shown by TLC and HPLC, and the radiochemical purity was greater than 97% as determined by these same methods.

Isolation and culture o f tracheal explants Animals were anesthetized using sodium pentobarbital (0.5 cc, 35 mg i.p.) and exsanguinated by severing the brachial arteries. Tracheae were removed, and trimmed of loose tissues, cut longitudinally, flattened, then placed epithelial side up on a stainless steel grid in an individual vial containing 1.2 ml of medium. The medium used was Dulbecco's Minimal Eagle (GIBCO, Grand Island, NY) supplemented with gentamycin ( 2 0 tag/ml) and buffered to pH 7.8 with sodium bicarbonate. [14C]AFBI was added in a volume of 12 ul of ethanol (final ethanol concentration 1%); control cultures received 12 ul ethanol containing no [14C]AFB~. Vials were incubated 24 h at 37°C in covered petri dishes (6 vials/dish) in an atmosphere of O2/COz (95:5) and a relative humidity of approx. 100%. At the end of the incubation period, the explants were removed from culture, and a thin (approx. 2 mm) slice of tissue was removed from approximately the midsection of the trachea, placed in modified Karnovsky's fixative [ 21] for later microscopic and autoradiographic analysis. The remaining portion of each trachea as well as the culture medium were separately placed in plastic vials, then frozen in liquid nitrogen and stored at - 8 0 ° C until analysis. DNA purification and analysis DNA was purified by a modification of the Marmur procedure [15]. Tracheae were homogenized in 1 ml of cold Merchant's solution (0.14 M NaC1, 1.47 mM KH2EDTA; pH 7.4) in a glass hand homogenizer. After centrifugation (1000 g) the pellet was resuspended in 0.75 ml 0.1 M NaC1, 50 mM EDTA (pH 7.2) then 0.75ml of a 2× lysis buffer was added (2% SDS, 12% sodium aminosalicylate, 2% NaC1, 12% 2-butanol) followed by 95 ul of pronase (56 PUK; Calbiochem, LaJolla, CA). The mixture was then incubated for 1 h at 37°C in a shaker bath. The lysate was then shaken for 1 h with 0.75 ml of 0.1 M Tris-saturated phenol and 0.75 ml of isoamylalcoholchloroform (IAC; 1 : 24). After centrifugation (1000 g, 20 min) the nucleic acids in the upper layer were extracted twice with equal vols. of IAC and then precipitated with 2 vols. of cold 95% ethanol with 0.3 M sodium acetate. The nucleic acids were then collected by centrifugation (3000 g, 5 min), washed and redissolved in 1.5 ml 0.1 M NaC1, 50 mM EDTA pH 7.2 buffer. After addition of ribonuclease (35 U RNase A, 475 U RNase T~, Sigma), the mixture was incubated 1 h at 37°C. Pronase (56 PUK) was then added and incubated 2 h at 37°C. Impurities in each DNA sample were then extracted with tris-saturated phenol and then with IAC until protein at the interface was no longer evident. The precipitated DNA was washed once in 95% ethanol, once in 75% ethanol, then redissolved in 2.0 ml H:O. Aliquots were analyzed for DNA by the Burton method [16], and specific activity determined by liquid scintillation (Beckman model LS 8000 counter) in Instagel cocktail (Packard, Downers Grove, IL). 119

Analysis of unbound AFB1 metabolites The culture medium was extracted with equal volumes of chloroform 3 times. The chloroform extracts were dried under nitrogen, dissolved in chloroform and aliquots spotted on plastic precoated silica gel 60 TLC plates (E Merck, Darmstadt, FRG). The plates were first developed in ethyl ether then in chloroform/acetone/isopropanol (85: 1 0 : 5 ) [17]. AFB1 metabolites were identified by their RF values relative to cochromatographed standards visible under long-wave UV light and associated radioactivities were determined by cutting o u t metabolite spots, then counting by liquid scintillation. The radioactivity present in the chloroform-extracted medium representing water soluble AFB~ metabolites was also determined.

Preparation of hamster $9 and mutagen assay Tracheas, removed as described above, were weighed and homogenized in 3 vols. of ice-cold sterile 0.15 M KC1 in an all-glass hand homogenizer. The homogenate was centrifuged at 9000 g for 10 min at 0°C. The supernatants ($9) were aliquoted into small plastic vials, frozen in liquid N2 and stored at - 8 0 ° C until the day of the assay. All operations were carried out at 4°C using sterile labware and cold, sterile solutions. $9 from hamster liver was prepared as above with cold 0.15 M KC1 before homogenization. Protein c o n t e n t of the $9 preparations were determined by the method of Lowry et al. [18]. A sensitive, modified Ames assay employing a microsuspension of $9, Salmonella typhimurium TA 98 (a gift of B.N. Ames, Berkeley, CA) and various doses of AFB~ was carried out as previously described [19] to compare the efficiency of tracheal $9 in activation of AFBI to bacterial mutagens relative to liver $9. Each assay plate received 50 ~g of tracheal or liver $9 protein.

Microscopic and autoradiographic analysis Fixed specimens from explant cultures were dehydrated in a graded series of ethanols and e m b e d d e d in glycol methacrylate. Two-micron thick sections were m o u n t e d on glass slides and dipped in Ilford L-4 emulsion ( 1 : 1 with water at 4°C), then exposed for 1 week in the dark at 4°C. The exposed slides were developed for 3 min in K o d a k D-19, rinsed and fixed in 15% sodium thiosulfate (Mallincrodt) then stained with toluidine blue. For electron microscopy, one saggital section from each dose was washed in Zettergist's wash [20] and postfixed in 1% OsO4 in Zettergist's solution for 2 h at 25°C. The tissues were washed and block-stained in 2% uranyl acetate overnight. The specimens were dehydrated in a graded series of ethanols followed by propylene oxide and e m b e d d e d in aryldite. One-micron thick sections were made to select regions that were over cartilage rings and had good cross-sectional orientation. These regions were trimmed of cartilagenous tissue and reembedded in beam capsules for ultrathin sectioning. Thin sections (approx. 90 nm) were made with a Sorvall MT2B ultramicrot o m e and a diamond knife and m o u n t e d on 150 mesh or single slotted grids (Pelco). Specimens were viewed and photographed with a Zeiss model EM-10 transmission electron microscope. 120

RESULTS

The amount of purified DNA isolated from pools of tracheal explants ranged from 20 to 124 pg. Binding of AFBl to tracheal DNA in culture was found to be linearly related to dose (Fig. 1) over a range of 0.1-1.0 I*M AFBl using a 24-h incubation period. Results of the comparative mutagenicity assays demonstrated the ability of S9 prepared from hamster trachea to activate AFBt to bacterial mutagens (Fig. 2a). However, the activity present in tracheal S9 was substantially less than that found in liver S9 (Fig. 2b); on the basis of revertants/nmole AFB,, as calculated by least squares linear regression. The liver/trachea activity ratio was approximately 70 : 1 (4.68 X lo4 f 1.83 X lo4 and 667 f 176 revertants/nmol AFBl for liver and trachea, respectively). The microsuspension assay employed was particularly well suited for testing tracheal S9 since it requires the addition of relatively low amounts of S9 protein as well as mutagen. Protein levels in tracheal S9 were extremely low (approx. 8.0 mg/ ml S9) which precluded the use of the standard plate incorporation assay to compare metabolic activities of tracheal and liver S9 preparations. Analysis of chloroform extracts of the culture medium from the explant cultures showed conversion of AFBl to AFL and AFQ1, as well as to polar derivatives (Table I). Metabolite formation occurred at all dose levels, but analysis of metabolites from the high dose (1.0 r.lM) cultures proved to be more accurately quantitated, and hence the results presented represent those cultures. AFL, a major metabolite of AFB1, was found to represent

a

E \

160

-

E

E a z E ,o

120-

o-

0.1

0.5

cont.

AFB,

I .o (pM)

Fig. 1. DNA binding at varying AFB, concentration in hamster tracheal explants. Values represent means (? range) of duplicate determinations of 5 pooled explants.

121

I000

I000

TRACHEAL

$9

800

LIVER

$9

.008

.012

800 G)

(,9 I-Z n~ w > w n~

t-z

600

600

rr hl > LU n-

400

O hl (D D Q Z

a W

¢D D O Z

200

400

200

0 0

I .2

I .4

I .6

I .8

~ I

nmoIs of A F B I per P L A T E

I 1.2

0 0

D04

.016

nmols of AFB I per PLATE

Fig. 2. Mutagenic activation efficiencies o f $9 prepared from hamster trachea (a) and liver (b) in a microsuspension mutagen assay using Salmonella typhimurium TA 98. Each assay plate received 50 ug o f tracheal or liver $9 protein. Data points represent means (± range) o f triplicate determinations. Background reversion values have been subtracted. Two such comparative experiments were carried out, o f which these results are representative.

about 16% of the total radioactivity recovered while AFQI accounted for only 2%. Unreacted AFB, accounted for the majority of the recovered radioactivity. Light microscopy {Fig. 3) showed that relatively little change occurred in TABLE I METABOLIC CONVERSION OF ['4C ] AFB, IN HAMSTER TRACHEAL EXPLANTS Metabolite

%a,b,c

Origin (polar)

16.8

AFQ, AFB,

2.10 (0.i0) 51.92 (6.15)

(1.76)

AFL

16.46 (2.88)

a Values expressed as percentage of total radioactivity recovered from the TLC chromatogram. b ± range of metabolite yields from supernatants o f 2 groups o f 5 pooled explant cultures. c AFB, concentration o f 1 ~M.

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Fig. 3. Light micrographs of toluidine blue stained 2 PM sections of hamster trachea after 24 h in culture with control (a), 0.1 PM (b), 0.5 PM (c) and 1.0 PM (d) AFB,. Note the swelling, vacuolation and accumulation of cytoplasmic dense droplets in non-ciliated cells that increases in intensity with increasing dose. At 1.0 PM (d) sloughed necrotic cells are present on the luminal surface. Ciliated cells appear relatively unchanged. X580.

123

the control explants. The 0.1 pm explants had more prominent non-ciliated cells with swollen cytoplasm. The explants in 0.5 and 1.0 PM AFBl had epithelial cells with cytoplasmic vacuoles containing dark staining spherical droplets. At the 1.0 r.lM concentration (Fig. 3d), some explants had occasional aggregates of sloughed cells on the epithelial surface and cytoplasmic droplets were more frequent. Autoradiographs showed a greater density of silver grains over the epithelium but some apparently non-selective labeling of submucosal tissues was evident. Epithelial grain density was dosedependent but the distribution of silver grains appeared to be non-specific with respect to cell type. The pattern of 14C binding within each cell appeared to be non-specific with the exception of apparent Increased grain densities overlying vacuoles containing dark droplets (data not shown). A

Fig. 4. Electron micrographs of hamster trachea after 24 h in culture with 0 (a) or 1.0 PM aflatoxin B,. The high dose epithelium has necrotic cells (N) and vacuolated and degenerate non-ciliated cells (arrows) containing electron dense cytoplasmic droplets and vacuolar structures. Ciliated cells have occasional electron dense cytoplasmic droplets but are otherwise unaffected. X2655.

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Ultrastructurally, control epithelium after 24 h in culture (Figs. 4a and 5a) resembled that from previously published reports of normal hamster trachea [12]. Non¢iliated cells contained abundant SER (Fig. 5a). Explants incubated with 0.1 pM AFB, had non-ciliated cells with dilated endoplasmic reticulum and occasional dilated vacuoles containing aggregates of lamellar membranous structures (Fig. 5b). At the 0.5 and 1.0 /aM concentrations, ciliated cells were relatively unaffected (Fig. 5d). Non-ciliated cells frequently contained membrane b o u n d round cytoplasmic aggregates of electron dense material and irregular arrays of membranous debris (Figs. 5c and d). These cells also had dilated endoplasmic reticulum (Fig. 5d) but mitochondria and nuclei remained relatively unaffected. Large poorly defined aggregates of membranous structures admixed with electron dense amorphous material were present occasionally in the 0.5/aM specimen and frequently in the 1.0 pM specimen (Figs. 5c and d). These structures were interpreted to represent necrotic cells. DISCUSSION

Although AFB, is primarily a hepatocarcinogen, this toxin has also been shown to prodLlce neoplasms in extrahepatic tissues [2]. Since high levels of AFB1 have been found in grain dusts and other respirable particles, activation by pulmonary MFOs resulting in carcinogenic initiation is a distinct potential. Recent studies have demonstrated that AFB, produces DNA adducts in respiratory cell cultures or explants similar to those found in liver DNA in vivo [22,23]. The purpose of our study was to further evaluate AFB, action in tracheal explants, with particular attention to the nature of DNA binding, metabolism and cellular pathology. The results clearly demonstrate the ability o f respiratory tissue to metabolize AFB, to an intermediate responsible for bacterial mutagenesis and alkylation of tracheal DNA. It can be seen from the comparative mutagenicity data, that on a mg protein basis, liver $9 is substantially more active in AFB, activation than tracheal $9. This trend may reflect differences in overall content of c y t o c h r o m e P-450 present in the 2 organs rather than absolute organ susceptibility to AFB, carcinogenesis. Organ specificity to chemical carcinogenesis is determined in large part by factors other than activation processes, including differences in fidelity of DNA repair, cell turnover, and detoxification capabilities. These parameters are not assessed by the bacterial mutagenesis and in the in vitro DNA binding studies. A direct comparison is complicated by the finding that in at least one rodent species, the rabbit, P-450 is highly concentrated in one cell t y p e in the distal lung and probably in the upper airways as compared to hepatocytes, where this enzyme system is weakly dispersed throughout the tissue [24]. In addition, since the metabolically active epithelial layer of hamster trachea contributes a small proportion to the total soluble protein in tracheal $9 (about 30% -- data not shown), the AFB,-activation efficiency of tracheal $9 relative to liver $9 may, in fact, be greater than reported. 125

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The major chloroform-extractable AFB1 metabolite formed by tracheal explants was AFL, a major hepatic metabolite whose production appears to correlate with tumor responsiveness [25,26,32]. Since AFL is a potent mutagen [27] and carcinogen [26], it is not thought to represent a detoxification product of AFB1. The presence of a substantial amount of labeled polar material in the chloroform extracts and in the resultant aqueous phases (data not shown), as well as the formation of AFQ,, demonstrates the ability of tracheal tissue to detoxify AFB,. Although the polar fractions were not analyzed, we interpret these to represent at least in part, sulfate, glucuronide and/or glutathione conjugates of AFB~. AFQ,, a hydroxylated metabolite of AFB~, is significantly less active biologically than the parent compound [27,28] and is considered to be a significant microsomal detoxification product [ 29,30]. Microscopic, ultrastructural and autoradiographic analysis of sections from each explant were performed to examine the distribution of label with respect to cell type and to assess the morphologic responses of airway epithelium to in vitro AFB1 exposure. The analyses indicate that AFB~ is bound within epithelial cells and causes dose-dependent degenerative changes primarily within non-ciliated epithelial cells. The susceptibility of nonciliated bronchiolar cells to toxicants is thought to be due to the high levels of cytochrome P-450 in these cells relative to that in other cell types [13]. Since non-ciliated bronchiolar cells in hamster contain abundant SER (the probable cellular location of cytochrome P-450) relative to ciliated cens [12], the selective necrosis of the morphologically similar non-ciliated epithelial cells due to AFB, suggests a correlation between SER content and cytochrome P-450 activity in large airways. The membrane-bound electron dense bodies in the cytoplasm of AFB~-treated non-ciliated cells resemble large multivesicular bodies and autophagosomes [33]. These structures are a non-specific response to sublethal injury and in this study may represent aggregation of chemically altered endoplasmic reticulum and denatured cellular proteins in autophagocytic vacuoles. The relatively dense autoradiographic grains in these structures suggest that those alterations were due to exposure to AFB, or its metabolites. Although greater autoradiographic grain density was observed over necrotic and segregated cellular debris, grain counts did not reveal significant differences in binding between cell types. This finding may be due to cell-tocell diffusion of AFBI and metabolites over the relatively long (24 h) exposure period {Harris, C.C., pets. comm.). Alternatively, nonspecific binding of AFB~ to cellular proteins of both cell types may be important. This interpretation implies that although proteins are a large cellular sink for AFB,, they are not a critical target resulting in cellular injury, especially in light of the cell-specific ultrastructural cellular damage observed. That AFB~ binding to protein does not directly relate to tissue injury has been previously reported [31]. Studies are currently underway to characterize the kinetics of AFB, uptake, particularly at early incubation intervals.

128

ACKNOWLEDGEMENTS

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