Cancer Letters, 62 (1992) 87 - 93 Elsevier Scientific Publishers Ireland
Carcinogen-DNA hepatocytes D.K. Monteith”
87 Ltd.
adducts in cultures of rat and human
and R.C. Guptab
’ Department tiue
of Biological Sciences, Indiana University-Purdue University at Fort Wayne Medicine and Environmental Health, and Graduate Center for Toxicology, Uniuersity
40506
and bDepartment of Kentucky,
of Preuen-
Lexington.
KY
(U.S.A.)
(Received 9 May 1991) (Revision received 18 October (Accepted 21 October 1991)
1991)
Summary Exposure to chemical carcinogens can often be identified by detection of DNA adduct lesions. Primary cultures of isolated rut and human hepatocytes were exposed to Z-acetylaminofluorene (AAfl, 4-aminobiphenyl (ABP), or benzo[a]pyrene (BP). The isolated DNA from the exposed cells was analyzed using the 32P-past-labeling assay. A greater total of carcinogen-DNA adducts, 2 - IZ-fold, were observed in human hepatocytes than rat hepatocytes at the same concentrations. The predominant DNA adducts for each carcinogen were the same between rat and human cells. The N-(deoxyguanosin-8-yI)-2-aminofluorene (dG-CB-AF) was the major AAF-DNA adduct. The N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the major ABP-DNA ad-
Correspondence to: D.K. maceuticals, 2800 Plymouth
Monteith. Parke-Davis PharRoad, Ann Arbor, Ml 48105,
duct. In the rat N2- ( 1 O/3-(7fl, &2,9a- trihydroxy- 7,8,9, IO-tetrahydrobenzo[a]pyrene)yl) deoxyguanosine (dG-N*-BP) and two unidentified adducts were nearly equivalent in amount, while the major BP-DNA adducts in the humans was the dG-N2-BP. The rat hepatocyte in vitro results are comparable to the predominant adducts found with rats exposed in vivo. The two different cultures of human hepatocytes demonstrated qualitative and quantitative differences in specific DNA adducts from rat hepatocytes. This study and others using human hepatocyte cultures demonstrate that this in vitro system can provide useful information for assessing human carcinogenic hazards.
Keywords: 2-acetylaminofluorene; benzo[a]pyrene, 4-aminobiphenyl; human; rat; hepatocytes; DNA adducts and 32P-postlabeling assay
U.S.A. AAF, 2-acetylaminofluorene; ABP, 4-aminobiphenyl; BP, benzo[a]pyrene; dG-C8-AF. N-(deoxyguanosinS-yl)-2-aminofluorene; dG-C%AAF. N-(deoxyguanosin-8-y& 2-acetylaminofluorene; dG-N*-AAF. 3-(deoxyguanosin-N2-yl)2-acetylaminofluorene; dG-C8-ABP, N-(deoxyguanosin-8-y& 4-aminobiphenyl; dG-N2-BP, N*- [ 10&(7P,8a,9wtrihydroxy7.8,9,10-tetrahydrobenzo[o]pyrene)yl]deoxyguanosine.
Abbreuiotions:
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and Published
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Introduction A variety of chemical carcinogens covalently bind to DNA to form chemical-DNA adducts. These adducts have been used as biomarkers of carcinogen exposure and the specific DNA
88
damage may contribute to mutagenesis, carcinogenesis or teratogenesis. Because of the differences in species responses to carcinogens, biomarkers such as DNA adducts may similarly vary in different species. Numerous papers discuss the detection of DNA adducts in human tissue exposed in vivo and in vitro [1,6,9,10,12,18,20,22,23,28,29]. As monitoring of peripheral blood lymphocyte DNA adducts in humans becomes more commonplace for assessing human exposure, the significance of these adducts with respect to target tissues becomes very important [10,12,20,22,23,28]. Animal models are an appropriate system in which to assess peripheral adducts with respect to target tissues. However, target tissues in animal models may not appropriately reflect target tissues in humans, or metabolic differences in the liver may reflect different activation of chemicals that affect other tissues. Therefore, investigations using normal human cells in vitro become important to evaluate the predictive value of animal models. Cultures of isolated parenchymal hepatocytes from different species have been used extensively in carcinogenesis research to investigate metabolism and DNA binding of carcinogens. The human hepatocyte culture system is ideal for studies of the mechanisms of genotoxic effects of chemicals in humans and risk assessment that would otherwise be conducted with non-human cells. Using the intact cell affords the investigation of the metabolism of a compound with a complete complement of enzymatic pathways. With an in vitro system, use of human cells avoids potential problems of interspecies extrapolation [13,14,16,17,21,24 - 261. This study compares the formation of chemical-DNA adducts in rat and human hepatocytes in primary culture with 2-acetylaminofluorene (AAF), 4-aminobiphenyl (ABP) or benzo[a]pyrene (BP). Methods Human hepatocytes were isolated from hepatic donor tissue. Case one was a 47-year-old male and case two was a 26-year-old female.
Normal hepatic tissue was perfused with collagenase as described [24]. Two separate experiments were performed using rat parenchyma1 hepatocytes isolated from two male Sprague - Dawley rats (250 g) obtained from Harlan Sprague - Dawley Laboratories (Indianapolis, IN) in a modified two-step collagenase perfusion [27]. The viability of the cells by trypan-blue exclusion were 80% for case one, 92% for case two, and >90% for rat hepatocytes. The hepatocytes were cultured on coilagencoated 100-mm plates with Eagle’s minimum essential media with non-essential amino acids (MEM) supplemented with insulin (0.1 mM), dexamethasone (0.1 PM), and 5% fetal bovine serum. Hepatocytes were allowed to attach to the culture substrate for 2 - 4 h, at which time the cultures were washed with serum-free MEM. Each dish contained approximately 8 x lo6 cells per plate and two plates per concentration. All experiments were done in duplicate. After cells had attached to collagen coated culture dishes, the medium (MEM) was replaced with serum-free medium supplemented with the carcinogen dissolved in DMSO (final concentration DMSO = 1%). Rat hepatocytes received 0, 0.1, 1.0, and 10 PM of AAF, ABP, or BP. Because of the limited number of cells, human hepatocytes received only a 10 PM concentration .of AAF, ABP, and BP. The cultures were left undisturbed for 24 h at 37OC in a 5% CO2 atmosphere. After incubation the medium was discarded and the cells rinsed with cold Hepes buffer and removed with a teflon scraper. The cells were centrifuged at 15 000 x g for 20 min. DNA was isolated from cells using a solvent extraction procedure as described [3,6]. DNA adducts were analyzed using a 32P-postlabeling assay [2] with butanol [5] or nuclease Pl [4] enrichment. Nuclease Pl treatment was used for BP treated cultures and a butanol extraction was used for AAF and ABP. Adduct levels were calculated by relative adduct labeling (RAL) which were translated into mol adducVpg DNA [5].
89
Results After 24 h of exposure to the carcinogens all hepatocyte cultures appeared to have the normal cuboidal morphology with no morphological signs of toxicity. The amount of
a.
CL Sham
Sham
AAF
d.
BpP
boAAF
C. ABP
C. ABP
b.
d.
BgP
autoradiograms of 32P-pastFig. 1. Representative labeling analysis of rat hepatocellular DNA following administration of: (a) control (DMSO); (b) 2-AAF; (c) 4-ABP; (d) BP to cells in culture. Circled areas, the locations of minor adducts, which are visible upon longer autoradiographic exposure. In each case 5 pg of DNA was analyzed. Solvents were as follows: Dl = 1 M sodium phosphate, (pH 6.0); D3 = 3.5 M lithium formate, 7 M urea (pH 3.5); D4 = 0.8 M lithium chloride, 0.5 M Tris-HCI, 7 M urea (pH 8.0). The major adduct for AAF was adduct 2 (dG-CS-AF). The adduct 4 (dGNs-AAF) was the second most abundant AAF adduct. The major ABP adduct was adduct 2 (dGC8ABP) and the major adducts for BP were adduct 3 (dG-N*-BP), adduct 4 (unidentified), and adduct 7 (derived from further metabolism of BP-trons-7,8-dihydrodiol). Maps with some of the major adducts are overexposed to display the minor adducts.
Fig. 2. Representative autoradiograms of 32P-pastlabeling analysis of human hepatocellular DNA following administration of: (a) control (DMSO); (b) 2-AAF; (c) 4-ABP; (d) BP to cells in culture. Circled areas, the locations of minor adducts, which are visible upon longer autoradiographic exposure. In each case 5 pg of DNA was analyzed. Solvents were the same as those used with the rat DNA. Adducts that have similar chromatographic migration positions are labeled with the same numbers between rats and humans.
DNA yield from human cells varied between 3.3 and 9.4 pg DNA/lo6 cells, while the yield from rat hepatocytes was 2.5 - 3.0 pg DNA/lo6 cells. Analysis of DNA indicated formation of multiple adducts in rat and human hepatocytes. These adducts were qualitatively similar between rat and human hepatocytes. Representative autoradiograms of the thin-layer chromatograms are presented in Figs. 1 and 2 for rat and human hepatocytes, respectively. The quantitative data for
90
Table 1. Carcinogen
Rat hepatocyte cont.
DNA adduct
DNA binding
(PM)
Adduct
2-AAF”
10 1.0 0.1
data
(amol adduct/pg
2
3
1830 zt 270 410 f 100 140 zt 52
BP
10e 1.0 0.1
5 270 f 50 30~ 6 ND
3
320 30 f 10 ND
10 1.0 0.1
4 20* 5 NDd 30 zt 25
Adduct 4-ABPb
DNA)
190 35* ND
2-AAFa
5
6
7
570 f 20 f 4*
90 10 1
580 f 60 20 l 10 3* 1
130 f 20 5* 2 ND
50 f 20 2* 1 ND
470 f 45* 6&
Adduct
HH2 Adduct
BP
HHl HH2
DNA adduct
1
1
114 1 207 577
100 15 1
1800 f 92 f 13*
Adducts
2, 8, and 9 were not detected.
experiments
performed
in duplicate
(n = 4).
represent
mean of duplicate
samples
DNA) 3
5
6
Total
7357 5136
98 53
37 9
83 32
7604 5255
2
3
4
5
Total
3544
63
ND’
65
3786
5102 18 684
4 612 1059
293 35 2
Adduct 6 was not detectable PM are adduct 1, 38; adduct 5, 50; adduct 6, 35;
2
2 and 3
“Adduct 4 was not detectable. bAdducts 6 and 7 were not detected. ‘ND, not detected.
Total
rat and human cells. Adducts were identified by cochromatography with appropriate reference adducts [2,4]. The dG-C8-AF adduct was the major AAF-DNA adduct (adduct 2).
data. Numbers
(amol adduct/pg
29 26 Adduct
4-ABPb
35 6
4
DNA binding
HHl HH2
2260 140 f 12*
1
3
hepatocyte
Carcinogen (10 PM)
Total
Adduct
specific adducts are presented in Tables I and II for rat and human hepatocytes, respectively. The predominant DNA adducts for each carcinogen were generally the same between
Human
2190 f 340 440 f 110 170 f 80
4
“Adduct 1 was not quantified (estimated value 18 f 5 at 10 PM). bAdducts 1, 5, 6, and 7 were not quantified (estimated values at 10 adduct 7, 55 amol/pg). ‘Adduct 1 was not quantified (estimated value 40 f 6 at 10 PM). dND, adduct not detected. eData from only single analyses, other data represent mean of two
Table II.
70 f 20 lo* 1 ND
2
1750 80 zt 25 12zt 6
Total
5 68 108
6 60 146
7
8
9
56 32
45 103
ND 317
Total 6150 21 026
The second most prevalent adduct of AAF was adduct 4 (dG-N2-AAF). The dG-C8-AAF adduct was not detected in either the human or rat hepatocytes. A previous study has demonstrated dG-C8-AAF to decrease with time in rat hepatocyte cultures [ll]. Recovery of the dG-CS-AAF adduct is in both the dinucleotide and mononucleotide forms which are present in approximately equal proportions. The butanol extraction procedure used in this study would not recover the dinucleotide [3,5,7,11]. The dG-C8-ABP adduct was the major ABPDNA adduct. The major BP adducts varied between rat and humans. In the rat, adduct 7, dG-N2-BP and one unidentified adduct (No. 4) were nearly equivalent in amount (Table I), while the major BP-DNA adduct in the humans was the dG-N2-BP (Table II). BP adduct 7 which was previously identified in rat liver after in vivo exposure and has been determined to be derived from the further metabolism of BP-trans-7,8-dihydrodiol [19]. The BP adducts 2 and 3 are partially resolved in human hepatocytes and adduct 3 (dG-N2-BP) was approximately 85% of the totals for the combined adduct 2 and 3 (Table II). In the rat, adduct 2 is not noted; however, its partial resolution from adduct 3 may be indicated by comparing panel d of Figs. 1 and 2. Adducts 2 and 4 in both the rat and the human hepatocytes may be diasteriomers or enantiomers of the dG-N2-BP adduct (Dipple, pers. commun.). Rat hepatocytes were exposed at concentrations of 0.1, 1 .O and 10 PM of each of the carcinogens. The rat hepatocytes demonstrated a correlation between increasing concentration and increasing adduct formation with each of the carcinogens. Altering the concentration of the carcinogen did not change which adducts were the predominant adducts. Discussion A comparison between different humans, and humans and rats is limited by the number of human cases; however, several general comparisons can be made considering these
individual humans. A greater total of carcinogen-DNA adducts, 2 - 12-fold, was observed in human hepatocytes than rat hepatocytes at the same concentrations. The differences between rats and humans may not be evident when averaged over a diverse human population; however, it does indicate that some individuals will demonstrate increased DNA binding of some carcinogens compared to laboratory animals. Most important, in the findings of this study were the differences in specific adducts. The quantities and types of specific adducts varied between rats and humans. Adduct 4 (dG-N2-AAF) of AAF exposed rat hepatocytes was not detected in human hepatocytes. This might suggest that human liver does not catalyze the sulfotransferase-mediated activation of N-hydroxy-2-acetylaminofluorene. Differences in specific adducts that are repaired could be related to differences in cellular DNA repair between rats and humans. Adducts 1, 2, 8 and 9 of BP exposed human hepatocytes were barely detected or not detected at all in rat hepatocytes. The relative proportions of specific adducts with respect to total adducts detected were comparable between human hepatocytes for BP and AAF exposures (Tables I and II). The absolute and relative amounts of the ABP adducts varied between rat and human hepatocytes. These data demonstrate qualitative and quantitative variations in adduct formations in isolated hepatocytes from rat and human tissue. The variations are most likely the result of metabolic differences demonstrated in human and rat hepatocytes previously [15,17,21], or possibly DNA repair differences. The metabolic and genotoxic differences would suggest potential carcinogenic differences between rats and humans. While this is a limited study, human hepatocytes can be useful models in the assessment of carcinogenic hazards for humans. Acknowledgments We would like to thank Ms. Marcia Boswell
92
and the Indiana University Surgical Transplant Program for their assistance in obtaining liver tissue and Ms. Karen Earley for technical assistance. This research was supported in part by an American Cancer Society Institutional Grant (IN-17), a Purdue Research Foundation XL Grant, and USPHS Grant CA30606 and EPA co-operative agreement CR-816185. Although, research was supported in part by the USEPA Co-operative Research Agreement CR816185, this manuscript has not been subjected to Agency review and therefore does not neccessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
K.L., Randerath, K., Phillips, D.H., Hewer, A., Santella, R.M., Young, T.L. and Perera, F.P. (1990) DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis.
13
11, 1229- 1231. Howard, P.C., Casciano, D.A., Beland, F.A. and Shaddock, J.G. (1981) The binding of N-hydroxy2-acetylaminofluorene to DNA and repair .of the adducts in primary rat hepatocyte cultures. Carcinogenesis, 2, 97 - 102. Liou, S.H., Jacobson-Kram, D., Poirier, M.C., Nguyen, D.. Strickland, P.T. and Tockman, M.S. (1989) Biological monitoring of fire fighters: sister chromatid exchange and polycyclic aromatic hydrocarbon-DNA adducts in peripheral blood cells. Cancer Res., 49, 4929- 4935. Monteith, D.K., Novotny, A., Michalopoulos. G. and
14
Strom, S.C. (1987) Metabolism of benzo(a)pyrene in primary cultures of human hepatocytes: dose-response over a four-log range. Carcinogenesis, 8, 983 - 988. Monteith, D.K., Michalopoulos, G. and Strom, S.C.
15
(1988) Metabolism of acetylaminofluorene in primary cultures of human hepatocytes: dose-response over a fourlog range. Carcinogenesis, 9. 1835- 1841. Monteith, D.K. and Strom, S.C. (1990) A comparison of
11
12
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Chacko, M. and Gupta, R.C. (1988) Evaluation of DNA damage in the oral mucosa of tobacco users and nonusers. Carcinogenesis, 9, 2309 - 2314. Gupta, R.C., Reddy, M.V. and Randerath, K. (1982) 32P-postlabeling analysis of non-radioactive aromatic adducts. Carcinogenesis, 3, carcinogen-DNA
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1081- 1092. Gupta, R.C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. U.S.A., 81,
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6943 - 6947. Gupta, R.C. and Dighe, N.R. (1984) Formation and removal of DNA adducts in rat liver treated with N-hydroxy derivatives of 2-AAF. Carcinogenesis, 5. 343 - 349.
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Gupta, R.C. (1985) Enhanced sensitivity of 32Ppostlabeling analysis of aromatic carcinogen: DNA adducts. Cancer Res., 45, 5656-5662 Gupta, R.C., Earley, K. and Sharma, S. (1988) Use of human peripheral blood lymphocytes to measure DNA binding capacity of chemical carcinogens. Proc. Natl. Acad. Sci. U.S.A., 85, 3513-3517. Gupta, R.C. (1988) 32P-adduct assay: short- and longterm persistence of AAF-DNA adducts and other applications of the assay. Cell Bioi. Toxicol., 4, 467 - 474. Gupta, R.C. and Earley, K. (1988) 32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 9, 1687 - 1693. Hatch, M.C., Warburton, D. and Santella, R.M. (1990) Polycyclic aromatic hydrocarbon-DNA adducts in spontaneously aborted fetal tissue. Carcinogenesis, 11, 1673 - 1675. Grzybowska, E., Chorazy. M.. Hemminki, K., Twardowska-Saucha, K., Srocrynski, J.W., Putman,
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the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and binding of 2-acetylaminofluorene. Cell Biol. Toxicol., 6, 269 - 284. Moore, C.J. and Gould, M.N. (1984) Metabolism of benzo(a)pyrene by cultured human hepatocytes from multiple donors. Carcinogenesis, 5, 1577 - 1582. Neis, J.M., Yap, S.H., Van Gemert, P.J.L., Roelofs, H.M.J. and Henderson, P.T.H. (1985) Mutagenicity of five arylamines after metabolic activation with isolated dog and human hepatocytes. Cancer Lett. 27, 53-60. Randerath. E., Miller, R.H., Mittal, D., Avitts, T.A., Dunsford, H.A. and Randerath, K. (1989) Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay. J. Natl. Cancer Inst., 81, 341- 347. Ross, J., Nelson, G., Klingerman, A., Erexson, G., Bryant, M.. Earley, K., Gupta, R. and Nesnow, S. (1990) Formation and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA. Cancer Res., 50, 5088- 5094. Rothman, N., Poirier, M.C., Baser, M.E., Hansen, J.A., Gentile, C., Bowman, E.D. and Strickland, P.T. (1990) Formation of polycylic aromatic hydrocarbon-DNA adducts in peripheral white blood cells during consumption of charcoal-broiled beef. Carcinogenesis, 11, 1241- 1243. Rudo, K., Meyers, W.C.. Dauterman, W. and Langenbath. R. (1987) Comparison of human and rat hepatocyte metabolism and mutagenic of activation 2-acetylaminofluorene. Cancer Res., 47, 5861- 5867. van Schooten, F.J., Hillebrand. M.J.X., van Leeuwen, F.E., Lutgerink, J.T., van Zanderwijk, N.. Jansen, H.M. and Kriek, E. (1990) Polycyclic aromatic hydrocarbonDNA adducts in lung tissue from lung cancer patients. Carcinogenesis, 11, 1677 - 1681.
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van Leeuwen, Hart, A.A.M.,
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Strom, S.C., Jirtle, R.L.. Jones, R.S.. Novicki, D.L., Rosenberg, M.R., Novotny, A., Irons, G., Mclain, J.R. and Michalopoulos, G. (1982) Isolation. culture, and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68. 771- 778. Strom, S.C., Novicki, D.L., Novotny, A., Jirtle, R.L. and Michalopoulos, G. (1983) Human hepatocytes-mediated mutagenesis and DNA repair activity. Carcinogenesis. 4, 683 - 686. Strom, S.C., Jirtle, R.L. and Michalopoulos, G. (1983) Genotoxic effects of 2-acetylaminofluorene on rat and
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human hepatocytes. Environ. Health Perspect. 49, 165- 170. Strom, S.C., Monteith. D.K., Manoharan. K. and NovotA. (1987) Genotoxicity studies with human ny, hepatocytes. In: The Isolated Hepatocyte: Use in Toxicology and Xenobiotic Transformation, pp. 265 - 280. Editors: E.J. Rauckman and G. Padilia. Academic Press, New York. Wiencke, J.K., McDowell, M.L. and Bodell, W.J. (1990) Molecular dosimetry of DNA adducts and sister chromatid exchanges in human lymphocytes treated with benzo(a)pyrene. Carcinogenesis. 11. 1497 - 1502. Wilson, V.L., Weston, A, Manchester, D.K., Trivers, G.E., Roberts, D.W., Kadlubar, F.F.. Wild, C.P., Montesano, R.. Willey, J.C. and Mann, D.L. (1989) Alkyl and aryl carcinogen adducts detected in human peripheral lung. Carcinogenesis, 10, 2149 - 2153.
Carcinogen-DNA hepatocytes D.K. Monteith”
87 Ltd.
adducts in cultures of rat and human
and R.C. Guptab
’ Department tiue
of Biological Sciences, Indiana University-Purdue University at Fort Wayne Medicine and Environmental Health, and Graduate Center for Toxicology, Uniuersity
40506
and bDepartment of Kentucky,
of Preuen-
Lexington.
KY
(U.S.A.)
(Received 9 May 1991) (Revision received 18 October (Accepted 21 October 1991)
1991)
Summary Exposure to chemical carcinogens can often be identified by detection of DNA adduct lesions. Primary cultures of isolated rut and human hepatocytes were exposed to Z-acetylaminofluorene (AAfl, 4-aminobiphenyl (ABP), or benzo[a]pyrene (BP). The isolated DNA from the exposed cells was analyzed using the 32P-past-labeling assay. A greater total of carcinogen-DNA adducts, 2 - IZ-fold, were observed in human hepatocytes than rat hepatocytes at the same concentrations. The predominant DNA adducts for each carcinogen were the same between rat and human cells. The N-(deoxyguanosin-8-yI)-2-aminofluorene (dG-CB-AF) was the major AAF-DNA adduct. The N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the major ABP-DNA ad-
Correspondence to: D.K. maceuticals, 2800 Plymouth
Monteith. Parke-Davis PharRoad, Ann Arbor, Ml 48105,
duct. In the rat N2- ( 1 O/3-(7fl, &2,9a- trihydroxy- 7,8,9, IO-tetrahydrobenzo[a]pyrene)yl) deoxyguanosine (dG-N*-BP) and two unidentified adducts were nearly equivalent in amount, while the major BP-DNA adducts in the humans was the dG-N2-BP. The rat hepatocyte in vitro results are comparable to the predominant adducts found with rats exposed in vivo. The two different cultures of human hepatocytes demonstrated qualitative and quantitative differences in specific DNA adducts from rat hepatocytes. This study and others using human hepatocyte cultures demonstrate that this in vitro system can provide useful information for assessing human carcinogenic hazards.
Keywords: 2-acetylaminofluorene; benzo[a]pyrene, 4-aminobiphenyl; human; rat; hepatocytes; DNA adducts and 32P-postlabeling assay
U.S.A. AAF, 2-acetylaminofluorene; ABP, 4-aminobiphenyl; BP, benzo[a]pyrene; dG-C8-AF. N-(deoxyguanosinS-yl)-2-aminofluorene; dG-C%AAF. N-(deoxyguanosin-8-y& 2-acetylaminofluorene; dG-N*-AAF. 3-(deoxyguanosin-N2-yl)2-acetylaminofluorene; dG-C8-ABP, N-(deoxyguanosin-8-y& 4-aminobiphenyl; dG-N2-BP, N*- [ 10&(7P,8a,9wtrihydroxy7.8,9,10-tetrahydrobenzo[o]pyrene)yl]deoxyguanosine.
Abbreuiotions:
Printed
and Published
in Ireland
Introduction A variety of chemical carcinogens covalently bind to DNA to form chemical-DNA adducts. These adducts have been used as biomarkers of carcinogen exposure and the specific DNA
88
damage may contribute to mutagenesis, carcinogenesis or teratogenesis. Because of the differences in species responses to carcinogens, biomarkers such as DNA adducts may similarly vary in different species. Numerous papers discuss the detection of DNA adducts in human tissue exposed in vivo and in vitro [1,6,9,10,12,18,20,22,23,28,29]. As monitoring of peripheral blood lymphocyte DNA adducts in humans becomes more commonplace for assessing human exposure, the significance of these adducts with respect to target tissues becomes very important [10,12,20,22,23,28]. Animal models are an appropriate system in which to assess peripheral adducts with respect to target tissues. However, target tissues in animal models may not appropriately reflect target tissues in humans, or metabolic differences in the liver may reflect different activation of chemicals that affect other tissues. Therefore, investigations using normal human cells in vitro become important to evaluate the predictive value of animal models. Cultures of isolated parenchymal hepatocytes from different species have been used extensively in carcinogenesis research to investigate metabolism and DNA binding of carcinogens. The human hepatocyte culture system is ideal for studies of the mechanisms of genotoxic effects of chemicals in humans and risk assessment that would otherwise be conducted with non-human cells. Using the intact cell affords the investigation of the metabolism of a compound with a complete complement of enzymatic pathways. With an in vitro system, use of human cells avoids potential problems of interspecies extrapolation [13,14,16,17,21,24 - 261. This study compares the formation of chemical-DNA adducts in rat and human hepatocytes in primary culture with 2-acetylaminofluorene (AAF), 4-aminobiphenyl (ABP) or benzo[a]pyrene (BP). Methods Human hepatocytes were isolated from hepatic donor tissue. Case one was a 47-year-old male and case two was a 26-year-old female.
Normal hepatic tissue was perfused with collagenase as described [24]. Two separate experiments were performed using rat parenchyma1 hepatocytes isolated from two male Sprague - Dawley rats (250 g) obtained from Harlan Sprague - Dawley Laboratories (Indianapolis, IN) in a modified two-step collagenase perfusion [27]. The viability of the cells by trypan-blue exclusion were 80% for case one, 92% for case two, and >90% for rat hepatocytes. The hepatocytes were cultured on coilagencoated 100-mm plates with Eagle’s minimum essential media with non-essential amino acids (MEM) supplemented with insulin (0.1 mM), dexamethasone (0.1 PM), and 5% fetal bovine serum. Hepatocytes were allowed to attach to the culture substrate for 2 - 4 h, at which time the cultures were washed with serum-free MEM. Each dish contained approximately 8 x lo6 cells per plate and two plates per concentration. All experiments were done in duplicate. After cells had attached to collagen coated culture dishes, the medium (MEM) was replaced with serum-free medium supplemented with the carcinogen dissolved in DMSO (final concentration DMSO = 1%). Rat hepatocytes received 0, 0.1, 1.0, and 10 PM of AAF, ABP, or BP. Because of the limited number of cells, human hepatocytes received only a 10 PM concentration .of AAF, ABP, and BP. The cultures were left undisturbed for 24 h at 37OC in a 5% CO2 atmosphere. After incubation the medium was discarded and the cells rinsed with cold Hepes buffer and removed with a teflon scraper. The cells were centrifuged at 15 000 x g for 20 min. DNA was isolated from cells using a solvent extraction procedure as described [3,6]. DNA adducts were analyzed using a 32P-postlabeling assay [2] with butanol [5] or nuclease Pl [4] enrichment. Nuclease Pl treatment was used for BP treated cultures and a butanol extraction was used for AAF and ABP. Adduct levels were calculated by relative adduct labeling (RAL) which were translated into mol adducVpg DNA [5].
89
Results After 24 h of exposure to the carcinogens all hepatocyte cultures appeared to have the normal cuboidal morphology with no morphological signs of toxicity. The amount of
a.
CL Sham
Sham
AAF
d.
BpP
boAAF
C. ABP
C. ABP
b.
d.
BgP
autoradiograms of 32P-pastFig. 1. Representative labeling analysis of rat hepatocellular DNA following administration of: (a) control (DMSO); (b) 2-AAF; (c) 4-ABP; (d) BP to cells in culture. Circled areas, the locations of minor adducts, which are visible upon longer autoradiographic exposure. In each case 5 pg of DNA was analyzed. Solvents were as follows: Dl = 1 M sodium phosphate, (pH 6.0); D3 = 3.5 M lithium formate, 7 M urea (pH 3.5); D4 = 0.8 M lithium chloride, 0.5 M Tris-HCI, 7 M urea (pH 8.0). The major adduct for AAF was adduct 2 (dG-CS-AF). The adduct 4 (dGNs-AAF) was the second most abundant AAF adduct. The major ABP adduct was adduct 2 (dGC8ABP) and the major adducts for BP were adduct 3 (dG-N*-BP), adduct 4 (unidentified), and adduct 7 (derived from further metabolism of BP-trons-7,8-dihydrodiol). Maps with some of the major adducts are overexposed to display the minor adducts.
Fig. 2. Representative autoradiograms of 32P-pastlabeling analysis of human hepatocellular DNA following administration of: (a) control (DMSO); (b) 2-AAF; (c) 4-ABP; (d) BP to cells in culture. Circled areas, the locations of minor adducts, which are visible upon longer autoradiographic exposure. In each case 5 pg of DNA was analyzed. Solvents were the same as those used with the rat DNA. Adducts that have similar chromatographic migration positions are labeled with the same numbers between rats and humans.
DNA yield from human cells varied between 3.3 and 9.4 pg DNA/lo6 cells, while the yield from rat hepatocytes was 2.5 - 3.0 pg DNA/lo6 cells. Analysis of DNA indicated formation of multiple adducts in rat and human hepatocytes. These adducts were qualitatively similar between rat and human hepatocytes. Representative autoradiograms of the thin-layer chromatograms are presented in Figs. 1 and 2 for rat and human hepatocytes, respectively. The quantitative data for
90
Table 1. Carcinogen
Rat hepatocyte cont.
DNA adduct
DNA binding
(PM)
Adduct
2-AAF”
10 1.0 0.1
data
(amol adduct/pg
2
3
1830 zt 270 410 f 100 140 zt 52
BP
10e 1.0 0.1
5 270 f 50 30~ 6 ND
3
320 30 f 10 ND
10 1.0 0.1
4 20* 5 NDd 30 zt 25
Adduct 4-ABPb
DNA)
190 35* ND
2-AAFa
5
6
7
570 f 20 f 4*
90 10 1
580 f 60 20 l 10 3* 1
130 f 20 5* 2 ND
50 f 20 2* 1 ND
470 f 45* 6&
Adduct
HH2 Adduct
BP
HHl HH2
DNA adduct
1
1
114 1 207 577
100 15 1
1800 f 92 f 13*
Adducts
2, 8, and 9 were not detected.
experiments
performed
in duplicate
(n = 4).
represent
mean of duplicate
samples
DNA) 3
5
6
Total
7357 5136
98 53
37 9
83 32
7604 5255
2
3
4
5
Total
3544
63
ND’
65
3786
5102 18 684
4 612 1059
293 35 2
Adduct 6 was not detectable PM are adduct 1, 38; adduct 5, 50; adduct 6, 35;
2
2 and 3
“Adduct 4 was not detectable. bAdducts 6 and 7 were not detected. ‘ND, not detected.
Total
rat and human cells. Adducts were identified by cochromatography with appropriate reference adducts [2,4]. The dG-C8-AF adduct was the major AAF-DNA adduct (adduct 2).
data. Numbers
(amol adduct/pg
29 26 Adduct
4-ABPb
35 6
4
DNA binding
HHl HH2
2260 140 f 12*
1
3
hepatocyte
Carcinogen (10 PM)
Total
Adduct
specific adducts are presented in Tables I and II for rat and human hepatocytes, respectively. The predominant DNA adducts for each carcinogen were generally the same between
Human
2190 f 340 440 f 110 170 f 80
4
“Adduct 1 was not quantified (estimated value 18 f 5 at 10 PM). bAdducts 1, 5, 6, and 7 were not quantified (estimated values at 10 adduct 7, 55 amol/pg). ‘Adduct 1 was not quantified (estimated value 40 f 6 at 10 PM). dND, adduct not detected. eData from only single analyses, other data represent mean of two
Table II.
70 f 20 lo* 1 ND
2
1750 80 zt 25 12zt 6
Total
5 68 108
6 60 146
7
8
9
56 32
45 103
ND 317
Total 6150 21 026
The second most prevalent adduct of AAF was adduct 4 (dG-N2-AAF). The dG-C8-AAF adduct was not detected in either the human or rat hepatocytes. A previous study has demonstrated dG-C8-AAF to decrease with time in rat hepatocyte cultures [ll]. Recovery of the dG-CS-AAF adduct is in both the dinucleotide and mononucleotide forms which are present in approximately equal proportions. The butanol extraction procedure used in this study would not recover the dinucleotide [3,5,7,11]. The dG-C8-ABP adduct was the major ABPDNA adduct. The major BP adducts varied between rat and humans. In the rat, adduct 7, dG-N2-BP and one unidentified adduct (No. 4) were nearly equivalent in amount (Table I), while the major BP-DNA adduct in the humans was the dG-N2-BP (Table II). BP adduct 7 which was previously identified in rat liver after in vivo exposure and has been determined to be derived from the further metabolism of BP-trans-7,8-dihydrodiol [19]. The BP adducts 2 and 3 are partially resolved in human hepatocytes and adduct 3 (dG-N2-BP) was approximately 85% of the totals for the combined adduct 2 and 3 (Table II). In the rat, adduct 2 is not noted; however, its partial resolution from adduct 3 may be indicated by comparing panel d of Figs. 1 and 2. Adducts 2 and 4 in both the rat and the human hepatocytes may be diasteriomers or enantiomers of the dG-N2-BP adduct (Dipple, pers. commun.). Rat hepatocytes were exposed at concentrations of 0.1, 1 .O and 10 PM of each of the carcinogens. The rat hepatocytes demonstrated a correlation between increasing concentration and increasing adduct formation with each of the carcinogens. Altering the concentration of the carcinogen did not change which adducts were the predominant adducts. Discussion A comparison between different humans, and humans and rats is limited by the number of human cases; however, several general comparisons can be made considering these
individual humans. A greater total of carcinogen-DNA adducts, 2 - 12-fold, was observed in human hepatocytes than rat hepatocytes at the same concentrations. The differences between rats and humans may not be evident when averaged over a diverse human population; however, it does indicate that some individuals will demonstrate increased DNA binding of some carcinogens compared to laboratory animals. Most important, in the findings of this study were the differences in specific adducts. The quantities and types of specific adducts varied between rats and humans. Adduct 4 (dG-N2-AAF) of AAF exposed rat hepatocytes was not detected in human hepatocytes. This might suggest that human liver does not catalyze the sulfotransferase-mediated activation of N-hydroxy-2-acetylaminofluorene. Differences in specific adducts that are repaired could be related to differences in cellular DNA repair between rats and humans. Adducts 1, 2, 8 and 9 of BP exposed human hepatocytes were barely detected or not detected at all in rat hepatocytes. The relative proportions of specific adducts with respect to total adducts detected were comparable between human hepatocytes for BP and AAF exposures (Tables I and II). The absolute and relative amounts of the ABP adducts varied between rat and human hepatocytes. These data demonstrate qualitative and quantitative variations in adduct formations in isolated hepatocytes from rat and human tissue. The variations are most likely the result of metabolic differences demonstrated in human and rat hepatocytes previously [15,17,21], or possibly DNA repair differences. The metabolic and genotoxic differences would suggest potential carcinogenic differences between rats and humans. While this is a limited study, human hepatocytes can be useful models in the assessment of carcinogenic hazards for humans. Acknowledgments We would like to thank Ms. Marcia Boswell
92
and the Indiana University Surgical Transplant Program for their assistance in obtaining liver tissue and Ms. Karen Earley for technical assistance. This research was supported in part by an American Cancer Society Institutional Grant (IN-17), a Purdue Research Foundation XL Grant, and USPHS Grant CA30606 and EPA co-operative agreement CR-816185. Although, research was supported in part by the USEPA Co-operative Research Agreement CR816185, this manuscript has not been subjected to Agency review and therefore does not neccessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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13
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Strom, S.C. (1987) Metabolism of benzo(a)pyrene in primary cultures of human hepatocytes: dose-response over a four-log range. Carcinogenesis, 8, 983 - 988. Monteith, D.K., Michalopoulos, G. and Strom, S.C.
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