ExpToxic Patho11996; 48: 544-547Gustav Fischer Verlag lena

Institute of Hygiene and Occupational Medicine, University of Essen, GermanyI Institute of ExperimentalPathology, Hannover Medical School, Germany

Significance of cell type specific formation and elimination ofDNA-adducts in respiratory tissues of hamster and rat inducedby alkylating chemical carcinogens*

F. SEILER, B. REHN, K. KAMIN0 1, M. EMURO, and 1. BRUCH

With 4 figures and 1 table

Received: December 1, 1995; Accepted: December 20, 1995

Address for correspondence: Dr. F. SEILER, Institute of Hygiene and OccupationalMedicine, University of Essen, Hufe­landstraBe 55,0-45122 Essen, Germany

Key words: DNA-adducts; Respiratory tissue; Carcinogens, alkylating; Tumour formation, species-specific; 0 6-ethyl­deoxyguanosine; Ethylnitrosourea; Diethylnitrosamine.

Introduction

Chemical carcinogens and various dusts have beenidentified as cancerogenic factors in the air we breath.Previous studies in different animal species have shownthat both the species and the test system (e. g. applicationmethod) have a great influence on the tumourigenic po­tency of the tested substances. Comparision of DNA ad­duct formation and repair at the cellular level among thecommonly-used animal models may help in understandingspecies-specific differences in tumour formation and alsohelp in selection of the most suitable test systems for riskassessment in humans. In a first set of experiments, lungand tracheal tissues of Syrian hamster and Wistar ratswere investigated for formation and repair of the DNA­adduct 0 6-ethyldeoxyguanosine (Os-Etdfluo) followingpulse alkylation with the monofunctional alkylating agentsethylnitrosourea (ENU) and diethylnitrosamine (DEN).ENU is a well characterized direct acting experimentalcarcinogen, whereas the metabolically activated DEN (byp450-monooxygenases) is found in trace amounts in en­vironment. 0 6-EtdGuo is repaired predominantly by therepair protein 0 6-alkylguanine-transferase (AGT) (PEGG,1990). Hamster and rat exhibit characteristic differencesin alkylating agent-induced tumours of the respiratory re­gion. Hamster tumours ocurred in high frequencies pre­dominantly in the trachea; rat tumours occur only in the

* Poster, presented at the 5th International Inhalation Sym­posium, Hannover, Germany, 20-24 February 1995.

544 ExpToxic Patho148 (1996)6

lung (for review: SCHMAHL et aI. 1980). Formation andelimination of DNA-adducts are quantified by a recentlydeveloped single cell immunofluorescence assay (lCA)(SEILERet al. 1993).

Material and methods

Male Syrian golden hamsters and Wistar rats were usedfor this investigation. The animals received a single intra­peritoneal injection of either diethylnitrosamine (DEN) orethylnitrosourea (ENU) a dose of 100 mglkg body weight.The animals were killed 0.5, 2, 6, 12, 24, 18 and 96 (101,rat) hours after exposure. Negative control animals weretreated with 0.9 % NaCI solution. Frozen sections (8 urn)were made from the selected organs and transfered onmicroscopic slides previously coated with ovalbumin.

leA: Immunofluorescence staining was performed ac­cording to the slightly modified ICA protocol (SEILERet al.1993). Cell samples were fixed in methanol and rehydratedin 2SSC. After treatment with RNAse A (200 ug/ml 2SSC)and RNAse T1 (50 units/ml 2SSC) for 1 h at 37 °C, cellswere washed in 0.14 M NaCl, and DNA was denatured byincubation with 70 mM NaOH/40 % EtOH in 0.14 M NaClfor 5 min at 0 DC. After washing with PBS/1 % BSA cellswere preincubated with PBS/20 % BSA for 20 min. 0 6-Etd­Guo specific monoclonal antibody EM 2-3 (EBERLE 1988)was diluted to 0.2 ug/ml PBS/l % BSA and slides weretreated for 16h at 4 °C, followed by intensive washing withPBS. Mouse anti rat-IgG F(abo-fragments conjugated withrhodamine isothiocyanate (TRITC) (Dianova, Hamburg, Ger­many), diluted to 2 ug/ml in PBS/l % BSA were added for45 min, 37 °C. After washing, nuclear DNA was counter­stained with the DNA-dye DAP (4.6-Diamidino-2-pheny­lindole) for 10 min, and mounted in PBS120 % Glycerol,

10080

- Pneumocytes II DEN

-0- Alveolarcells ENU

604020oo

80,----------------------,

120100

~Rat ENU

+ Rat DEN

71(.Hamster ENU

• Hamster DEN

80604020

280,-----------------_i'c~240

~iii!200

~i160()

!g120;:..::ge();:U 408-Ul

Time after pulse exposure (h) Time after pulse exposure (h)

Fig. 1. Kinetics of formation and eliminationof 06-EtGuafrom the DNA of hamster and rat trachea epithelial cellsafter pulse exposurewith 100mg END or 100mg DENlkgbody weight. Each point represents the average value for100individual cells.

Fig. 2. Kinetics of formation and eliminationof 06-EtGuafromthe DNAof hamsteralveolarcellsafterpulseexposurewith 100mg END or 100mg DEN / kg body weight. Eachpoint represents the average value for 100individual cells.

10 % Elvanol, pH 8.2 containing 0.03 M DTE to reducefading.

Quantification of DNAalkylation products: For visua­lization of nuclear fluorescence, a Zeiss Axiovert fluores­cence microscope with a HBO 100 W mercury lamp andZeiss standardfilter combinations wasused.For quantifica­tion, fluorescence images were recorded by a high resolu­tion CCD-camera (Hammamatsu, Japan) and fed into amultiparameter image analysisprogram(AHRENS ACAS,Bargteheide, Germany). This program enables quantifica­tion of both antibody-fluorescence signal and DNA-fluore­scenceof the same cell. Good separation of the objects andthe recognition of unmodified or slightly modified nucleiwas obtained by combining the DNA-fluorescence imageand the immunofluorescence image.DNA contentsand ad­duct-fluorescence were correlated and corrected. The stai­ning intensitywas expressedas the averagefluorescence ofabout 100nuclei per measurement.

elimination rate of only 50 % after 96 hours. In the ratonly 75 % of the peak hamster adducts were reached andthese were nearly completely eliminated after 101 hoursdue to the efficient repair capacity of rat epithelial cells.Interestingly in a few epithelial cells adducts persisted forlonger than 101 hours (fig. 4, white bars), obviously a re­pair deficient subpopulation. Such cells have been postu­lated to be present in different organs, but have not beenshown yet because non single cell techniques, e. g. post­labeling, HPLC, GC-MS or the competitive RIA requirethe isolation of bulk DNA and consequently will showonly overall levels of DNA-adducts.

Table 1. 06-EtGua specific nuclear staining in trachea andlung of the Syrianhamster after pulse treatment with DEN.

++++, very strong staining; +++, strong staining; ++, mo­derate staining; +, weak staining; -, no staining.

++++ Terminalbronchiole

++ Epithelium +++

Lung

Lung parenchymaSmall arteriesandveinsPneumocyte Type IPneumocytes Type II ++Septal cellsCapillaryendothelium

Smoothmuscle

+

CiliatedcolumnarepitheliumDucts ofthe tracheal glandsHyalinecartilageof the +tracheal ringTrachealismusclePerichondrium

Trachea

Substantial formation of 06-EtGua-DNA adducts afterDEN-exposure was observed in the trachea of both ratand hamster, predominantly concentrated in the epithe­lium (tab. 1.). Large differences exist in the repair capa­city of hamster and rat epithelial cells (fig. 1.). In the rat6 hours after END-pulse, 90 % of 06-EtGua-adducts hadbeen removed from nuclear DNA, whereas in the hamsterhigh amounts of DNA-adducts persistent for more than96 hours. After DEN exposure similar results could beobserved. Due to the continous metabolic activation,DNA-adduct level in both species reached a peak at24 hours. In the hamster elimination was slow, with an

Results

Trachea

Exp Toxic Pathol 48 (1996)6 545

Fig.3. Visualization of 0 6-EtGua residues in cyrostat sections of hamster trachea exposed to DEN. Micrographs (1) Phasecontrast; (2) Fluorescence of DNA stained with DAPI; (3) Fluorescence (immunostaining with anti-(06-EtdGuo) MabEM 2-3 and TRITC labeled anti-lg second antibodies. A: 4 h after exposure to DEN; B: 96 h after exposure to DEN ; C:Control animal. Magnification: 200x, E: Epithelium.

Fig.4. Visualization of Os-Etflua residues in cyrostat sections of rat trachea exposed to DEN . Micrographs (1) Phase con­trast; (2) Fluorescence of DNA stained with DAPI; (3) Fluorescence (immunostaining with anti-(06-EtdGuo) Mab EM 2-3and TRITC labeled anti-lg second antibodies. A: 4 h after exposure to DEN; B: 101 h after exposure to DEN; C: Controlanimal. Magnification: 400 x, E: Epithelium.

546 Exp Toxic Pathol 48 (1996) 6

Lung

Till now only the results from hamster lung are available.After DEN exposure staining was observed only in theepithelium of the bronchial system and in Type II pneu­mocytes (tab. 1). The repair capacity in both cell typeswas low, comparabel with trachea (fig. 1.) and other al­veolar cells (fig. 2.).

Discussion

To date a full explanation for species-specificity in tu­mour induction is not available. This is in part due to thelack of information about the cell progenitors for tumou­rigenesis. For these cells, the balance between metabolicactivation of carcinogenic agents and their ability to eli­minate produced DNA-adducts is important. Our experi­ments have shown that the single cell immunocytologicalassay (lCA) is a suitable tool to fill this gap. In the pre­sent experiments DNA ethylation and repair in the respi­ratory system of rat and hamster was determined. Bothspecies show cell type specific formation of DNA-adducts.In the trachea the two species have no significant diffe­rences in formation of DNA-adducts, but large differen­ces in repair capacity of target cells. These findings are ingood correlation with chemically induced tumour forma­tion, not only for alkylating compounds, but also for otherP 450 metabolized chemicals. Investigations will be ex-

panded to (l) further species and their common strains,(2) inhalation studies with dusts with and without adsor­bed carcinogens and (3) to various other relevant DNA­adducts, e. g. PAH-DNA-adducts and DNA-adducts in­duced by oxygen-radicals that are eliminated by excisionrepair mechanisms.Acknowledgements: Thanks are due to K. HEISE for herskilfull help in the processing of the rat trachea sections.The antibody has been kindly provided by Dr. M. F. RA­JEWSKY, Institute of Cell Biology, University of Essen. Thiswork has been supported by the Minister fur Wirtschaft,Mittelstand und Technologie, Nordrhein-Westfalen, Ger­many, Study Group for Dust Suppression and Silicosis Pre­vention.

References

EBERLE G: Ph. D. Dissertation 1989; University of Essen,Essen, Germany.

PEGG AE: Mammalian O'<alkylguanine-Dbl.A alkyltrans­ferase: regulation and importance in response to alky­lating carcinogens and therapeutic agents. Cancer Res1990;50: 6119-6129.

SEILER F, KIRSTEIN U, EBERLE E, et al.: Quantification ofspecific DNA O-alkylation products in individual cellsby monoclonal antibodies and digital imaging of inten­sified nuclear fluorescence. Carcinogenesis 1993; 9:1907-1913.

SCHMAHL D, HABS H: Carcinogenicity of N-Nitroso Com­pounds. Oncology 1980; 37: 237-242.

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