(CANCER RESEARCH 48, 7146-7149, December 15, 1988]

Gene-specific Differences in the Aflatoxin BI Adduction of Chicken Erythrocyte

Chromatin1,3GenevièveP. Delcuve,1 Richard Moyer,2 George Bailey,2 and James R. Davie

Department of Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada RÕE OWÌ¡J.R. D., G. P. D.J, and the Department of FoodScience and Technology; Oregon State University, Corvallis, Oregon 97331 [R. M., G. B.J

ABSTRACT

Mature and immature chicken erythrocyte nuclei were treated withactivated aflatoxin H, (2,3-dichloroaflatoxin H,), producing covalenti}1bound DNA adducts. This reaction produces alkali-labile sites in theDNA which can be identified by using a variation of the Maxam-Gilbertsequencing procedure. We determined the aflatoxin H, accessibility ofdefined regions of the erythroid genome by using different specific probesand monitoring the disappearance of similar-sized fragments generatedby restriction enzyme digestion. The genes studied were the erythroid-specific /3-globin and histone H5 genes, which are potentially active inmature erythroid nuclei and transcriptionally active in immature eryth-rocytes, and the vitellogenin and ovalbumin genes, which are both transcriptionally inactive in these cells. The /?-globin and histone H5 geneswere more accessible than the repressed vitellogenin and ovalbumin genesto aflatoxin I), modification in mature and immature erythroid chromatin.Micrococcal nuclease was used to probe the nucleosomal organization ofactive (0-globin and histone H5) and repressed (vitellogenin and ovalbumin) genes in chicken erythrocytes. The vitellogenin and ovalbumin genesshow a canonical nucleosome repeat pattern in mature and immaturechicken erythrocyte nuclei. In contrast, the /3-globinand histone H5 geneslack a distinct nucleosomal repeat pattern in these cells. These resultssupport the hypothesis that transcriptionally active genes are preferentially accessible to carcinogen modification because of their disruptedchromatin structure.

INTRODUCTION

i4 is a mycotoxin produced by certain strains of the

fungus Aspergillus flavus found as a naturally occurring foodcontaminant that has been implicated in the etiology of somehuman hepatic cancers (1,2). AFBi is one of the most potentprocarcinogens known. Activation of AFBr is believed to proceed via epoxidation of the 2,3-double bond, forming the AFB,-2,3-epoxide. AFBi can be activated in vitro by oxidation with amild oxidant like chloroperoxybenzoic acid. The synthetic analogue, 2,3-dichloroaflatoxin B,, is a good model for the reactivity, mutagenicity, and carcinogenicity of the 2,3-epoxide (3).The major initial DNA adduct formed by metabolically orchemically activated AFBi is jY-7-guanyl adduct, 2,3-dihydro-2-(JV7-guanyl)-3-hydroxyaflatoxin B, (4, 5).

The organization of chromatin in the nucleus is known toplay an important role in the functional aspects of gene regulation. It is now clearly established that genes which are expressed or have the potential to be expressed are in an alteredchromatin conformation which is sensitive to DNase I (6-8).For example, in the immature and mature chicken erythrocyte,the chromatin of the 0-globin and histone H5 genes is sensitiveto DNase I digestion, while the chromatin of transcriptionallyrepressed genes (ovalbumin and vitellogenin) is not (6, 9).Sensitivity to DNase I suggests an open or exposed chromatin

Received 5/19/88; revised 8/30/88; accepted 9/8/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by the National Cancer Institute of Canada.¡Supported by Grants ES03850. ES00210, and ES00541 from the USPHS.* MRC Scholar. To whom requests for reprints should be addressed.*The abbreviations used are: AFBi, aflatoxin B,; AFBi-Cl¡,2,3-dichloroafla

toxin B,; DH sites, DNase I hypersensitive sites; RSB. 10 mM Tris-HCl, pH 7.5,10 min NaCI, 3 mM MgCl2.

structure which would be accessible to the transcription machinery. The repressed class of gene chromatin structures maybe effectively invisible to the elements involved in transcrip-tional regulation.

Within the DNase I-sensitive domains are regions which aredevoid of nucleosomes and hypersensitive to DNase I digestion.These DH sites are found at the 5' and/or 3' ends of genes and

are the binding sites for sequence-specific DNA-binding proteins (7, 8). The chicken erythrocyte /3-globin and histone H5genes have DH sites located at the 5' and 3' ends of the gene

(Fig. l;Refs. 9-11).DNA is thought to be the critical macromolecular target in

chemical carcinogenesis. Since alterations in chromatin structure can determine the accessibility of specific DNA sequencesto a variety of agents, it is conceivable that some chromatinstructures are more vulnerable to attack by chemical carcinogens than are others (12, 13). Expressed genes or genes havingthe potential to be expressed could be more susceptible toaflatoxin B, adduction because of their open chromatin structure, leading to alterations in gene expression and/or to agreater extent of mutagenesis in the expressed gene loci (14).In this study, we determined whether 2,3-dichloroaflatoxin BIpreferentially attacks the chromatin of single-copy, transcriptionally active genes in chicken erythroid nuclei. This systemwas chosen because DNA replication does not occur in theterminally differentiated erythrocyte (15). Since newly replicated chromatin has an "open" configuration (16, 17), it would

presumably complicate the investigation of the relationshipbetween transcribed genes and AFBi adduction. Previously,aflatoxin BI was shown to preferentially modify the multicopy,ribosomal gene sequence (13). In this report, we demonstratethat the chromatin of the j3-globin and histone HS genes ispreferentially accessible to AFB, modification.

MATERIALS AND METHODS

Isolation and Digestion of Nuclei. Mature and immature chickenerythrocytes were obtained from adult White Leghorn chickens andanemic chickens (18), respectively, as previously described (19). Nucleiwere isolated as described in Ref. 19 and were resuspended to aconcentration of 50 A26ounits per ml in Buffer A (l M hexylene glycol,10 mM piperazine-/VyV'-bis(2-ethanesulfonic acid), pH 7.0, 2 mM

MgCl2, 30 mM sodium butyrate, 1% (v/v) thiodiglycol, and 1 mMphenylmethylsulfonyl fluoride) to which was added CaCl2 to 1 mM. Tothe nuclei suspension, micrococcal nuclease (Pharmacia) was added to25 A26ounits per ml, and the nuclei were digested for 30 min at 37°C.

The digestion was terminated by placing the suspension on ice andadding [ethylene bis(oxyethylenenitrilo]tetraacetic acid to 10 mM.

2,3-Dicloroaflatoxin H, Treatment of Erythroid Nuclei. AI B, (Õ..wassynthesized as previously described (3) except that the AFBi-Cl2 waschromatographed on a microPorasil column (Waters) with 2.5% acetone in méthylènechloride. Mature or immature chicken erythrocytenuclei were resuspended in RSB to a concentration of 4 A26ounits perml (200 Mg/ml). Various amounts of AFBi-Cl2 (0.01, 0.05, or 0.1 fig)were added to the nuclear suspension which was incubated at either 37or 0°Cfor 5 min. The nuclei were collected by centrifugation and

resuspended in RSB. This step was repeated.DNA Preparation and Restriction Endonuclease Digestions. The mi

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Fig. 1. Restriction maps of the chicken .>'

globin and histone HS genes. The open andclosed boxes indicate the position of the intronsand exons, respectively. The horizontal arrowshows the direction of transcription. The vertical arrows mark the DNase I hypersensitivesites. The DNA probes used in this study areshown.

AFLATOX1N B, ADDUCTION OF ACTIVE GENES

GLOBIN

CBG 13

1kb

H5

chV2.5 B/H

clear pellet was resuspended in RSB (pH 6.8). DNA was prepared asdescribed (19) except that the pronase digestion was for only 3.5 h at37°C,and it was digested with the appropriate restriction endonucle-

ase(s) (see Fig. 2). After restriction endonuclease digestions, DNA washeated at 90°Cfor 30 min in 1 M piperidine as described by Muench et

al. (20), electrophoresed on 1% alkaline agarose gels (21), and transferred to nitrocellulose. Hybridizations to 32P-labeled probes were done

as described in Ref. 19. The cloned DNA probes used were: pCBG13,DNA sequence of the adult chicken /3-globin gene which was acquiredfrom H. Martinson (Ref. 22; see Fig. 1). pVTG412, obtained from H.Weintraub, recognizes the 5' region of the chicken vitellogenin gene

(23). pchV 2.5 B/H from A. Ruiz-Carrillo is a 2.5-kilobase pair fragment of the chicken histone H5 gene (Ref. 24; see Fig. 1). pOV12 isthe chicken ovalbumin gene received from M-J. Tsai (25).

RESULTS

Accessibilities in Chromatin of Various Genes to ActivatedAflatoxin BI. Mature chicken erythrocyte nuclei were treatedwith different amounts of AFB,-C12 at 37°Cfor 5 min. The

DNA was isolated at slightly acidic pH in order to stabilize theAFBi-N7-Gua lesion (26) and digested with restriction endo-nuclease(s), BamHl/Hindlll or EcoRl. The AFB,-N7-Gua ad-duct is alkali labile, and a modification of the Maxam-Gilbertprocedure (i.e., treatment with piperidine) for DNA sequenceanalysis leads to breakage of the DNA fragment at the site ofthe modified residue (20, 27). The single-stranded DNA fragments were electrophoretically separated on 1% alkaline aga-rose gels, transferred to nitrocellulose, and hybridized to theindicated 32P-labeled DNA sequence (Fig. 2). Note that only

one guanine has to be modified to effect the elimination of the

EcoRl BamHI _ HinDIII

H5 GLOBIN

1 2 3 1 2 3

-2.5kb

. 2.1kb

Fig. 2. Aflatoxin B, accessibility of specific erythroid genes. Mature chickenerythroid nuclei were incubated with different amounts of AFBi-Ch (Lane 1,control; Lane 2, 0.01 ng/200 ng DNA; Lane 3, 0.10 <zg/200 (ig DNA) for 5 minat 37'C. The isolated DNA was digested with the indicated restriction endonucle

ase, treated with piperidine, and resolved on a 1% alkaline agarose gel. DNAfragments were visualized with ethidium bromide (DNA) or transferred to nitrocellulose and hybridized to the indicated 32P-labeled probe: ovalbumin (OVAL),histone H5 (HS). or 0-globin (GLOBIN).

restriction fragment in question. The gene probes used in thisstudy detected the following similar-sized restriction fragments:ovalbumin, 1.8- and 2.4-kilobase EcoRl fragments; vitellogenin,1.8- and 1.9-kilobase BamHl-Hindlll fragments; histone H5, a2.5-kilobase BamHl-Hindlll fragment; and /3-globin, a 2.1-kilobase BamHl-Hindlll fragment. Visual inspection of theautoradiograms suggests that the 2.1-kilobase ß-globinand 2.5-kilobase histone H5 DNA fragments are more sensitive toAFBi-Q2 attack than is the 2.4-kilobase ovalbumin DNA fragment (or vitellogenin DNA fragments, not shown). At a levelof 0.01 Mg/ml of AFB,-C12 the intensities of the 2.5-kilobasehistone H5 and 2.1-kilobase /3-globin restriction fragments isless than that of the respective fragments of untreated DNA. Incontrast, the intensities of the 2.4- and 1.8-kilobase ovalbuminDNA fragments are similar in both the untreated and treatedDNA samples. At higher levels of AFB,-C12 (0.1 Mg/ml), thereis a slight anodal shift in the DNA fragment pattern (DNA,Fig. 2), indicating that a greater amount of AFBi modificationhad occurred. At this level of AFBi modification, the majorityof the gene fragments studied were modified, as indicated bythe loss of the respective restriction fragments, including the2.4-kilobase ovalbumin DNA fragment. These results demonstrate that the chromatin of these genes has different accessibilities to AFBi-Ch, with potentially active gene chromatinbeing more accessible than repressed gene chromatin to AFBimodification.

The level of aflatoxin BI adduction of several genes in matureand immature chicken erythrocyte chromatin was quantified.The experiments were performed similarly to those describedabove except that the nuclei were treated with AFBi-Q2 at 0°C

for 5 min. The DNA was restricted with BamHI and Hindlll,treated with piperidine, and electrophoretically resolved on a1% alkaline agarose gel. The DNA fragments were transferredto nitrocellulose and hybridized with the indicated DNA sequence. The lanes of the autoradiogram were scanned, and theintegrated peak intensities were determined for the restrictionfragments of interest. The ratio of the intensities of the DNAfragments of AFBi-modified DNA to that of untreated DNAwas calculated and plotted versus the amount of AFBi-Cl2added. Fig. 3 shows that in both mature and immature erythroidnuclei, the 2.1-kilobase /3-globin and 2.5-kilobase histone H5DNA fragments are more readily modified than the 1.9-kilobasevitellogenin DNA fragment. The /3-globin and histone H5 genesare more accessible to AFBi modification in immature erythroidchromatin than in mature erythroid chromatin. Note that at aratio of 0.10 /¿gAFB,-C12 to 200 ng of DNA, 27% of the histoneH5 restriction fragment (31 % for 0-globin) remains in modifiedimmature erythroid DNA while 52% of this fragment (62% for

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AFLATOXIN B, ADDUCTION OF ACTIVE GENES

MATURE IMMATURE

100

90

80

i70

£60

S 50

£ 40

È30

K 20

IO

O05 01

AFE, Clj CONCENTRATION

lug /ml) lug/ml!

Fig. 3. Aflatoxin accessibility of specific genes in mature or immature chickenerythrocyte nuclei. Mature or immature chicken erythrocyte nuclei were incubatedwith different amounts of AFB|-C12 for 5 min at O'C. The isolated DNA was

digested with /(«mill and lliml\\\. treated with piperidine, resolved on a 1%alkaline agarose gel, and hybridized to a "P-labeled probe containing the DNAsequences of either /3-globin (D), histone H5 (•),or vitellogenin (A). The lanes ofthe autoradiograms were scanned, and the integrated peak intensities were determined for each restriction fragment. Percentage of hybridization of the intensityof the restriction fragment from the AFBi-CI2-treated nuclei versus the intensityof the restriction fragment from the untreated nuclei was calculated and plottedagainst the concentration of AFBi-Ch used.

DNA OVAL VTG H5 GLOBIN

Ml Ml Ml Ml Ml

II "

Fig. 4. Nucleosomal organization of specific genes in chicken erythroid nuclei.Nuclei isolated from mature (M) or immature (/) chicken erythrocyte nuclei weredigested with micrococca) nuclease. The isolated DNA fragments were electro-phoretically resolved on 1% agarose gels, transferred to nitrocellulose, and hybridized to the indicated ' '!' hilvk-d probe. The DNA fragments were visualized

with ethidium bromide (DNA). The DNA sequence of the probes used wereovalbumin (OVAL), vitellogenin ( VTG), histone H5 (A/5), and /3-globin (GLOBIN).

/3-globin) is left in treated mature erythroid. This is a representative result of several experiments (5 for mature and 2 forimmature cells).

Chromatin Structure of Transcriptionally Active and InactiveChicken Erythroid Genes. Mature and immature chicken erythroid nuclei were digested with micrococcal nuclease, and theDNA fragments were electrophoretically resolved on a 1%agarose gel. Fig. 4 (DNA) shows a typical nucleosome repeatpattern of chicken erythrocyte chromâtin. The DNA was transferred to nitrocellulose and hybridized to labeled DNA sequences of either transcript ionally active or inactive genes. Thetranscriptionally inactive ovalbumin and vitellogenin DNA sequences exhibit a canonical nucleosome array. However, theerythroid-specific 0-globin and histone H5 DNA sequences,which are potentially active in mature erythroid cells and activein immature cells, show a nondiscrete continuum of DNAlengths in both cell types (Fig. 4).

DISCUSSION

In this report, we demonstrate that the transcriptionallyactive and potentially active, single-copy, 0-globin and histone

H5 genes have an altered chromatin structure which is preferentially accessible to AFBi-Cb. The nondiscrete nucleosomalpattern is thought to be a result of an altered nucleosomal andhigher order chromatin structure. This alteration in chromatinstructure may be due to several alterations in the chromatin ofthese genes, including elevated levels of the modified histonespecies (e.g., acetylated and ubiquitinated histone species) whichmodify nucleosome structure (19, 28, 29), DH sites whichdisrupt higher order structure (30), and torsional stress whichmay disrupt both nucleosomal and higher order structure (31).

The DH sites are regions of chromatin which are devoid ofnucleosomes. Thus, these regions may be considered as longlinker regions. Since AFB, is located preferentially in the linkerDNA regions (32), it is conceivable that the DH sites of the ß-globin and histone H5 genes (see Fig. 1) would be preferentiallymodified. If such a reaction was occurring, we would expect tosee the generation of bands shorter than the restriction fragment(see Refs. 9 and 10 for examples). However, such bands werenot observed. This result suggests that the DH sites of the ß-globin and histone H5 genes are not hypersensitive to AFBi-Cl: attack. DH sites have unpaired bases and react with thepossible carcinogen, bromoacetaldehyde, which has a preference for unpaired DNA bases (8, 33). Since AFBi reacts poorlywith guanines that are in single-stranded DNA (34), it is notsurprising that DH sites are not preferred targets of AFBi.

The transcriptionally active, 0-globin and histone H5 genes,were preferentially accessible to AFBi-Cb modification compared to repressed genes, ovalbumin and vitellogenin. Sinceboth of the expressed genes were preferentially adducted, thisargues against fortuitous sequence-specific attack of AFB] (e.g.,in a gene enriched for GGG sequences) being responsible forthe greater sensitivity of these sequences to AFBi-Cl2 modification (20, 34, 35). Rather, these results suggests that thegreater sensitivity of the two active DNA sequences is due totheir more open chromatin structure.

The chromatin of the |8-globin and histone H5 genes wasmore accessible to AFBi-Cb modification in immature compared to mature erythroid nuclei. This suggests that the transcriptionally active state of these genes is more vulnerable toAFBi-Ch modification than the potentially active chromatinstate. We5 have observed that the chromatin of the /3-globin and

histone H 5 genes is in two states, one of which is soluble aspolynucleosomes at physiological ionic strength and the otherwhich has an insoluble character similar to that reported forthe globin genes in murine erythroleukemia cells (36). In immature chicken erythroid nuclei, approximately 50% of thechromatin of the /3-globin and histone H5 genes is in aninsoluble form, while 25% of these erythroid-specific genes isin an insoluble state in mature cells. It remains to be determinedwhether these two chromatin states are equally accessible toAFBi modification. It is noteworthy that the DNase I-sensitive,/3-globin and histone H5 genes, which are both expressed athigh rates, are located at the borders of condensed chromatinmasses along interchromatin channels which communicate withthe nuclear periphery (37). It is possible that the nuclear localization of these genes makes them accessible targets for chemical carcinogens.

The demonstration that chromatin state can influence genetarget access to carcinogens has important implications inunderstanding mechanisms of carcinogenesis. For example,AFBi is a well known hepatocarcinogen in the rainbow trout,but has never been observed to induce kidney tumors in over20 years of tumor studies in our laboratory (38), even at doses

5 G. P. Delcuve and J. R. Davie, manuscript in preparation.

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AFLATOXIN B, ADDUCTION OF ACTIVE GENES

above those which produce 100% liver cancer incidence. Despitethis lack of response, overall genomic AFBi-DNA adduction inkidney is 7-21% that found in liver of juvenile trout (39), andappears to be even higher in AFBi-exposed trout embryos.6 A

possible explanation is that the target protooncogene exists ina regional chromatin configuration which is protective in kidney, but accessible to AFBi in liver. In this regard it is interesting that yV-methyl-W-nitro-yV-nitrosoguanidine induces tumorsin kidney as well as liver in trout (38), because such smallalkylating agents may be less influenced by local chromatinstructure.7 We are currently examining the accessibility of thetrout Ki-ras protooncogene to yV-methyl-W-nitro-yV-nitroso-guanidine and AFBi in trout kidney and liver. Size differencesin attacking electrophile may also underly the recent observation that, at equivalent levels of overall genomic O6 methyla-tion, 7V-nitrosomethylbenzylamine and ./V-nitrosodimethyla-mine do not initiate foci equally (40). Similarly, the cell cycle-dependent initiation of hepatocarcinogenesis in rats bymethyl(acetoxymethyl)nitrosamine, which peaks during S-phase (41), may in part enhance target gene access as chromatinunfolds locally during replication. These hypotheses are readilytestable. Although there are some circumstances under whichoverall genomic damage level can provide a useful estimate oftumor induction (42, 43), it is likely to be a misleading indexwhen comparing risk among various organs, or for structurallyrelated carcinogens.

REFERENCES

1. Peers, F. G., and Linsell, C. A. Dietary aflatoxins and liver cancer—apopulation based study in Kenya. Br. J. Cancer, 27:473-487, 1973.

2. Shank, R. C., Bhamarapravati, N., Gordon, J. E., and Wogan, G. N. Dietaryaflatoxins and human cancer. IV. Incidence of primary liver cancer in twomunicipal populations in Thailand. Food Cosmet. Toxicol., 10: 171-179,1972.

3. Swenson, D. H., Miller, J. A., and Miller, E. C. The reactivity and carcino-genicity of aflatoxin Bi-2,3-dichloride, a model for the putative 2,3-oxidemetabolite of aflatoxin B,. Cancer Res., 35: 3811-3823, 1975.

4. Essigmann, J. M., Cray, R. G., Nadzan, A. M., Busby, W. F., Jr., Reinhold,V. N., Buchi, G., and Wogan, G. N. Structural identification of the majoradduci formed by aflatoxin B, in vitro. Proc. Nati. Acad. Sci. USA, 74:1870-1874, 1977.

5. Lin, J. K., Miller, J. A., and Miller, E. C. 2,3-Dihydro-2-(guan-7-yl)-3-hydroxy-aflatoxin B,, a major acid hydrolysis product of aflatoxin Bi-DNAor -ribosomal RNA adducts formed in hepatic microsome-mediated reactionsand in rat liver in vivo. Cancer Res., 37:4430-4438, 1977.

6. Reeves, R. Transcriptionally active chromatin. Biochem. Biophys. Acta, 782:343-393, 1984.

7. Yaniv, M., and Cereghini, S. Structure of transcriptionally active chromatin.Crit. Rev. Biochem., 21:1-26, 1986.

8. Eissenberg, J. C., Cartwright, I. L., Thomas, G. H., and Elgin, S. C. R.Selected topics in chromatin structure. Annu. Rev. Genet., 19: 485-536,1985.

9. Renaud, J., and Ruiz-Carrillo, A. Fine analysis of the active H5 genechromatin of chicken erythrocyte cells at different stages of differentiation.J. Mol. Biol., 189: 217-226, 1986.

10. McGhee, J. D., Wood, W. I., Dolan, M., Engel, J. D., and Felsenfeld, G. A200 base pair region at the 5' end of the chicken adult .(-niobin gene isaccessible to nuclease digestion. Cell, 27:45-55, 1981.

11. Emerson, B. M., Nickol, J. M., Jackson, P. D., and Felsenfeld, G. Analysisof the tissue-specific enhancer at the 3' end of the chicken adult /3-globingene. Proc. Nati. Acad. Sci. USA, 84: 4786-4790, 1987.

12. Arrand, J. E., and Murray, A. M. Benzypyrene groups bind preferentially tothe DNA of active chromatin in human lung cells. Nucleic Acids Res., 10:1547-1555, 1982.

13. Irvin, T. R., and Wogan, G. N. Quantitation of aflatoxin B, adduction withinthe ribosomal RNA gene sequences of rat liver DNA. Proc. Nati. Acad. Sci.USA,«/: 664-668, 1984.

14. McMahon, G., Hanson, L., Lee, J., and Wogan, G. N. Identification of anactivated c-Ki-ros oncogene in rat liver tumors induced by aflatoxin B,. Proc.Nati. Acad. Sci. USA, 83:9418-9422, 1986.

15. Williams, A. F. DNA synthesis in purified populations of avian erythroidcells. J. Cell Sci., 10: 27-46, 1972.

6 Unpublished results.7 R. Moyer, G. Bailey, and K. E. van Holde, manuscript in preparation.

16. Scale, R. L. Nucleosomes associated with newly replicated DNA have analtered conformation. Proc. Nati. Acad. Sci. USA, 75: 2717-2721, 1978.

17. Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L.Dual nature of newly replicated chromatin, evidence for nucleosomal andnon-nucleosomal DNA at the site of native replication forks. J. Biol. Chem.,256: 11880-11886, 1981.

18. Zhang, D., and Nelson, D. A. Histone acetylation in chicken erythrocytes:rates of acetylation and evidence that histones in both active and potentiallyactive chromatin are rapidly modified. Biochem. J., 250: 233-240, 1988.

19. Ridsdale, J. A., and Davie, J. R. Chicken erythrocyte polynucleosomes whichare soluble at physiological ionic strength and contain linker histones arehighly enriched in /3-globin gene sequences. Nucleic Acids Res., 15: 1081-1096, 1987.

20. Muench, K. F., Misra, R. P., and Humagun, M. Z. Sequence specificity inaflatoxin Bi-DNA interactions. Proc. Nati. Acad. Sci. USA, 80:6-10, 1983.

21. Maniatis, T., Fritsch, E. F., and Sambrook, J. In: Molecular Cloning, aLaboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982.

22. Villeponteau, B., Landes, G. M., Pankratz, M. J., and Martinson, H. G. Thechicken ßglobin gene region: delineation of transcription units and developmental regulation of interspersed DNA repeats. J. Biol. Chem., 257:11015-11023, 1982.

23. Burch, J. B. E., and Weintraub, H. Temporal order of chromatin structuralchanges associated with activation of the major chicken vitellogenin gene.Cell, 33:65-76, 1983.

24. Ruiz-Carrillo, A., Affolter, M., and Renaud, J. Genomic organization of thegenes coding for the six main histones of the chicken: complete sequence ofthe H5 gene. J. Mol. Biol., 770:843-859, 1983.

25. Woo, S. L. C., Dugaiczyk, A., Tsai, M-J., Lai, E. C., Catterall, J. F., andO'Malley, B. W. The ovalbumin gene: cloning of the natural gene. Proc.Nati. Acad. Sci. USA, 75: 3688-3692, 1978.

26. Groopman, J. D., Croy, R. G., and Wogan, G. N. In vitro reactions ofaflatoxin B,-adducted DNA. Proc. Nati. Acad. Sci. USA, 78: 5445-5449,1981.

27. Maxam, A. M., and Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol., 65:499-560, 1980.

28. Alonso, W. R., Ferris, R. C., Zhang, D., and Nelson, D. A. Chickenerythrocyte /3-globin chromatin: enhanced solubility is a direct consequenceof induced histone hyperacetylation. Nucleic Acids Res., 15: 9325-9337,1987.

29. Oliva, R., Bazett-Jones, D., Mezquita, C., and Dixon, G. H. Factors affectingnucleosome dissassembly by protamines in vitro: histone hyperacetylationand chromatin structure, time dependence, and the size of the sperm nuclearproteins. J. Biol. Chem., 262:17016-17025, 1987.

30. Caplan, A., Kimura, T., Gould, H., and Allan, J. Perturbation of chromatinstructure in the region of the adult /3-globin gene in chicken erythrocytechromatin. J. Mol. Biol., 193: 57-70, 1987.

31. Villeponteau, B., Lundell, M., and Martinson, H. Torsional stress promotesthe DNAase I sensitivity of active genes. Cell, 39:469-478, 1984.

32. Bailey, G. S., Nixon, J. E., Hendricks, J. D., Sinnhuber, R. O., and VanHolde, K. E. Carcinogen aflatoxin B, is located preferentially in internucleo-somal deoxyribonucleic acid following exposure in vivo in rainbow trout.Biochemistry, 19: 5836-5842, 1980.

33. Kohwi-Shigematsu, T., Gelinas, R., and Weintraub, H. Detection of analtered DNA conformation at specific sites in chromatin and supercoiledDNA. Proc. Nati. Acad. Sci. USA, 80:4389-4393, 1983.

34. Misra, R. P., Muench, K. F., and Humayun, M. Z. Covalent and noncovalentinteractions of aflatoxin with defined deoxyribonucleic acid sequences. Biochemistry, 22: 3351-3359, 1983.

35. Marien, K., Moyer, R., Loveland, P., van Holde, K., and Bailey, G. Comparative binding and sequence interaction specificities of aflatoxin Bi, aflatoxi-col, aflatoxin M, and aflatoxicol M, with purified DNA. J. Biol. Chem., 262:7455-7462,1987.

36. Cohen, R. G., and Sheffery, M. Nucleosome disruption precedes transcriptionand is largely limited to the transcribed domain of globin genes in murineerythroleukemia cells. J. Mol. Biol., 182: 109-129, 1985.

37. Hutchison, N., and Weintraub, H. Localization of DNAase I-sensitive sequences to specific regions of interphase nuclei. Cell, 43:471-482, 1985.

38. Bailey, G. S., Hendricks, J. D., Nixon, J. E., and Pawlowski, N. E. Thesensitivity of rainbow trout and other fish to carcinogens. Drug Metab. Rev.,15:725-750, 1984.

39. Bailey, G. S., Williams, D. E., Wilcox, J. S., Loveland, P. M., Coulombe, R.A., and Hendricks, J. D. Aflatoxin B, carcinogenesis and its relation to DNAadduci formation and adduci persistence in sensitive and resistant salmonidfish. Carcinogenesis (Lond.), in press, 1988.

40. Silinskes, K. C., Zucker, P. F., and Archer, M. C. Formation of O'-methyl-

guanine in rat liver DNA by nitrosamines does not predict initiation ofpreneoplastic foci. Carcinogenesis (Lond.), 6: 773-775, 1985.

41. Kaufman, W. K., Rice, J. M., Wank, M. L., Devor, D., and Kaufman, D. G.Cell cycle-dependent initiation of hepatocarcinogenesis in rats bymethyl(acetoxymethyl)nitrosamine. Cancer Res., 47: 1263-1266, 1987.

42. Lutz, W. K. In vivo covalent DNA binding as a quantitative indicator in theprocess of chemical carcinogenesis. Mutât.Res., 65: 289-356, 1979.

43. Dashwood, R. H., Arbogast, D. N., Fong, A. T., Hendricks, J. D., and Bailey,G. S. Mechanisms of anti-carcinogenesis by indole-3-carbinol; detailed invivo DNA binding dose-response studies after dietary administration withaflatoxin Bi. Carcinogenesis (Lond.), 9:427-432, 1988.

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1988;48:7146-7149. Cancer Res   Genevieve P. Delcuve, Richard Moyer, George Bailey, et al.   Chicken Erythrocyte Chromatin

Adduction of1Gene-specific Differences in the Aflatoxin B

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