Mutation Research, 272 (1992) 139-144 139 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1161/92/$05.00

MUTENV 08840

Mutation of V79 cells by N-dialkylnitrosamines after activation by hamster pancreas duct cells

T e r e n c e L a w s o n and Caro l K o l a r

Eppley Institute for Research on Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA

(Received 31 December 1991) (Revision received 31 March 1992)

(Accepted 29 April 1992)

Keywords: Duct epithelium; Nitrosamine; Pancreas; V79 cells; Hamster pancreas duct cells

Summary

Pancreas duct epithelial cells (DEC), isolated from hamsters and cultured for up to 25 days, were able to metabolize N-nitrosobis(2-oxopropyl)amine (BOP) to species that were mutagenic in V79 cells. There was no decline in the nitrosamine-activating ability of DEC over the period of observation (25 d). DEC activated N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosodiethylamine (DEN), N-nitroso- dimethylamine (DMN) and N-nitrosomethyl(2-oxopropyl)amine (MOP) and BOP in the same assay, although the mutation frequencies for BHP, DEN and DMN were barely different from that for the controls (4 + 1 mutants/106 cells). The mutation frequencies for a dose of 0.1 mM were BHP, 2 + 1; BOP, 113 + 7; DEN, 8 + 1; DMN, 5 + 2; and MOP, 18 + 3 (mutants/106 cells; means + SE). When hepatocytes were used the mutation frequencies were BHP; 3 + 1; BOP, 60 + 3; DEN, 8 + 2; DMN, 8 + 2; and MOP, 121 + 10. BOP was toxic to the DEC at doses above 0.1 mM. Experiments in which co-factors were omitted from the medium suggested that an isoform(s) of the cytochrome P-450 IliA family was involved, directly or indirectly, in BOP activation.

Supported by NIH grant CA43646, AICR grant 90All, NCI Cancer Laboratory Core grant CA36727 and ACS grant SIG 16.

Correspondence: Dr. Terence Lawson, Eppley Institute for Research in Cancer and Allied Diseases, University of Ne- braska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6805, USA. Tel. (402) 559-7475; Fax (402) 559-4651.

Abbrevmtions: BHP, N-nitrosobis(2-hydroxypropyl)amine [CAS No. 53609-64-6]; BOP, N-nitrosobis(2-oxopropyl)amine [CAS No. 60599-38-4]; DEN, N-nitrosodiethylamine [CAS No. 55-18-5]; DEX, dexamethasone [CAS No. 50-02-2]; DMN, N-nitrosodimethylamine [CAS No. 62-75-9]; MOP, N- nitrosomethyl(2-oxopropyl)amine [CAS No. 55984-51-5]; SDM, secondary duct medium; WE, Williams medium E.

Pancreatic ductular adenocarcinomas (PDA) are induced in Syrian hamsters by/3-hydroxy or -keto substituted dialkylnitrosamines related to N-nitrosodi-n-propylamine (Lawson et al., 1991). PDA are believed to develop from epithelial cells of the duct network (Pour et al., 1981) (DEC). DEC can be isolated and cultured (Heimann and Githens, 1991) allowing their response to carcino- gens to be measured. Carcinogens involved in the hamster /PDA model require metabolic activa- tion so it was important to measure the ability of DEC to activate chemicals that are carcinogenic to the pancreas. This paper describes the ability of hamster DEC to generate mutagens from di- alkyl-nitrosamine using the mutation of V79 cells

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to resistance to 6-thioguanine (TG) as the marker of mutagenicity (van Zeeland and Simons, 1976). There is a good correlation between carcino- geniticy of pancreas carcinogens and mutagenic- ity in V79 cells (Langenbach et al., 1980). V79 ceils are deficient in or have very low levels of the enzymes believed to be involved in dialkylni- trosamine metabolism (McGregor, 1991) and must be co-cultured with cells that can perform the metabolism, e.g. hepatocytes (Langenbach et al., 1980) or other cells (Cheng and Li, 1985). N- Nitrosobis(2-hydroxypropyl)amine (BHP), N- nitrosobis(2-oxopropyl)amine (BOP), N-nitroso- diethylamine (DEN), N-nitrosodimethylamine (DMN) and N-nitrosomethyl(2-oxopropyl)amine (MOP) were used. BHP, BOP and MOP induced PDA in hamsters (Pour et al., 1981). They are mutagenic in V79 cells as are DEN and DMN (Langenbach et al., 1980). BHP and MOP are metabolites of BOP in vivo (Lawson et al., 1981). DEN induces ductular hyperplasia in the hamster pancreas, which is claimed to represent PDA initiating activity (Amanuma et al., 1991). It is an attractive candidate as a PDA inducer since the persistence of O4-ethylthymine (T 4-Et) in the liver correlated with its carcinogenicity in that tissue (Dyroff et al., 1986) and there is evidence that BOP acts through a lesion other than O6-methyl - guanine (Lijinsky, 1991; Lawson, 1992), which we presume, but do not know, to be T 4Me. The ability of DEC and hepatocytes to activate BHP, BOP, DEN, DMN and MOP to mutagenic species was measured.

Materials and methods

Animals. Male Syrian hamsters (8 weeks old; 105-115 g) (Unei[SYR]) were used. Some were fed a high fat diet (Donnelly et al., 1987) for 7 d before use.

Cells. DEC, from pooled pancreases, were grown in secondary duct medium (SDM). A ham- ster pancreas produced six 60-mm dishes of DEC, at confluence. V79 cells were grown in Williams medium E (plus fetal bovine serum (FBS) (5%; v/v)) (WE).

Chemicals. BHP and DME/F12 were ob- tained from Sigma, St. Louis, MO, USA; BOP from Ash-Stevens Chemicals, Detroit, MI, USA; DEN and DMN from Aldrich, Milwaukee, WI, USA; WE and FBS from BRL-Gibco, Gaithers- burg, MD, USA; MOP (Pour et al., 1980) was synthesized.

Mutagenicity. Mutagenicity was measured as TG resistance (van Zeeland and Simons, 1976). DEC activation: ( i)V79 cells (2 X 106) were seeded in 6-well plates. 24 h later DEC that had been in secondary culture for at least 4 d on 404-P filters (Schleicher and Schuell, Keene, NH, USA), were transferred to the V79 cells by invert- ing the filter so that the DEC and V79 ceils were in contact and the mutagen added. The extent of DEC-V79 cell contact is not known but it is assumed not to be as extensive as that between V79 cells and hepatocytes in suspension. Using DEC filters allows them to be retrieved and used for other analyses. (ii) In a second assay DEC were cultured in SDM for 4 d when they were transferred to variants of SDM, (a) SDM, (b) SDM-dexamethasone (DEX), (c)SDM-insulin (INS), and (d) SDM-DEX and INS, and were maintained in these media for 7 d when they were used in the mutagenicity assay. Hepatocyte activation: (i) V79 cells were seeded in 25-cc T- flasks. Hepatocytes (Williams et al., 1980) were added 24 h later and were co-cultured with V79 cells in WE (Lawson et al., 1990). (ii) In a second experiment the effects of WE and SDM on hepa- tocyte activating ability were compared using BOP (1 mM) as the mutagen. Exposure to BOP was for 16 h.

Statistical analyses. Data were analyzed by one-way analysis of variance (ANOVA) (Gad and Weill, 1986).

Results

Co-culture of DEC and V79 cells. The DEC used in the co-culture procedures were main- tained on filters. Attempts were made to obtain DEC-V79 co-cultures by adding DEC as single- cell suspensions to V79 cells growing on collagen. DEC did not attach to the V79 monolayer and

TABLE 1

MUTAGENICITY IN V79 CELLS OF BOP (0.1 mM) ACTI- VATED BY HAMSTER DEC

Days in 2nd culture Mutants/106 cells a,b,c

8 82 + 5 (23) 14 103 + 7 (51) 21 113 +- 7 (48)

a Mean +- SE (n = 8); b Absolute survival plating efficiencies (%) in parentheses; c Background mutation frequency was 4+1 mutants/106 cells.

only attached to the flasks or plates where there were no V79 cells growing and they appeared to "pull away" from the V79 cells. V79 cells, as single-cell suspensions, would not attach to DEC growing on filters. In this case we believe that it is due to the polarity of the DEC on filters. We observed cilia on the apical surface on DEC on filters and it appears that the V79 cells will not attach to this (apical) surface, the one presented to them.

Mutagenicity of BOP activated by DEC. Mu- tation frequencies (mutants/106 cells) for BOP (0.1 mM) activated by hamster DEC that had been in secondary culture for 8, 14 and 21 d were 82±5 , 1 0 3 ± 7 and 113±7 (mean±SE, n = 8 ) (Table 1). They are not statistically different (measured by ANOVA). The background muta- tion frequency was 4 + 1 mutants/106 surviving cells. In Tables 1-5 "n" refers to the number of plates used in that part of the mutagenicity assay in which the number of mutants is measured (Langenbach et al., 1980). The dose of BOP (0.1 mM) was the highest that could be used without excessive lethality to the DEC.

Mutagenicity of BHP, BOP, DEN, DMN and MOP activated by DEC and hepatocytes. Muta- tion frequencies for BHP, BOP, DEN, DMN and MOP (0.1 mM) activated by DEC or hepatocytes are shown in Table 2. DEC were grown in SDM and hepatocytes in WE. When DEC were used mutation frequencies were BHP, 2 _+ 1; BOP, 113 ± 7; DEN, 8 ± 1; DMN, 5 ± 2; and MOP, 18 ± 3 (mean ± SE, n = 16). When hepatocytes were used mutation frequencies were BHP, 3 ± 1; BOP,

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TABLE 2

MUTAGENICITY IN V79 CELLS OF BHP, BOP, DEN, DMN AND MOP (0.1 mM) ACTIVATED BY HAMSTER DEC a OR HEPATOCYTES

Mutants/106 cells b,c

Hepatocytes d DEC

Control e 3_+ 1 (43) 4_+ 1 (67) BHP 3 +- 1 (69) 2 + 1 (36) BOP 60+ 3 (65) 113+-7 (31) DEN 8_+ 2 (52) 8 + 1 (43) DMN 8+- 2 (63) 5 +- 2 (33) MOP 121 _+ 10 (69) 18+-3 (55)

a DEC were in 2nd culture for 14 d; b Mean +- SE (n = 16); Absolute survival plating efficiencies (%) in parentheses;

d Hepatocytes were freshly isolated; e Cells plus V79 cells, no mutagen.

60 + 3; DEN, 8 + 2; DMN, 8 + 2; and MOP, 121 + 10.

Mutagenicity of BOP activated by hepatocytes cultured in SDM or WE. The mutation frequen- cies for BOP (1 mM) activated by hepatocytes that had been cultured for 1 or 3 d in WE or SDM were 173 + 12 (WE, 1 d), 218 + 23 (WE, 3 d), 199 _+ 13 (SDM, 1 d) and 180 _ 19 (SDM, 3 d). There were no statistical differences (measured by ANOVA) between days 1 and 3 in either group or between the two media (Table 3).

Mutagenicity of BOP (0.1 mM) activated by homogenates of duct tissue and hepatocytes. BOP (0.1 mM) was activated by homogenates of freshly isolated duct tissue and hepatocytes from the same hamsters so that a comparison could be made under similar conditions. Tissues from hamsters fed a normal diet and those fed a high

TABLE 3

MUTAGENICITY IN V79 CELLS OF BOP (1 mM) ACTI- VATED BY HEPATOCYTES GROWN IN SDM OR WE

D c Mutants/106 cells a,b

WE SDM

1 173+- 12 (28) 199+- 13 (22) 3 218 + 23 (21) 180 + 19 (18)

a Mean+_SE (n = 8); b Absolute survival plating efficiencies (%) in parentheses; c Days after isolation.

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fat diet (Donnelly et al., 1987) were used. Muta- tion frequencies are shown (Table 4). Duct tissue was more efficient than hepatocytes. Tissues from hamsters fed the normal diet produced 16.4 + 1.6 (duct) and 3.2 + 0.5 (hepatocytes) (mutants/106 cells/mg protein) while those from hamsters fed a high fat diet produced 37.9_+ 2.1 (duct) and 1.6 _+ 0.1 (hepatocytes).

Mutagenicity of BOP (0.1 mM) activated by DEC cultured in SDM + and - DEX and INS. Mutation frequencies for BOP (0.1 mM) acti- vated by DEC that had been in secondary culture for 8 d in variants of SDM were, (complete) 5 1 + 4 (mean+SE; n = 8 ) , ( - D E X ) 2 4 + 4 , ( - I N S ) 45 + 3, and ( - D E X and INS) 25 + 8 (Table 5).

Discussion

Hamster DEC cultured up to 25 d (4 d in primary and 21 d in secondary culture) metabo- lized BOP to species that mutated V79 cells to TG resistance. Pancreas duct-tissue homogenates more efficiently metabolize BOP to mutagenic species than acinar or hepatocyte homogenates (Lawson et al., 1990). This result (duct > hepatocyte) is confirmed in the present (Table 4) and in a related study (Lawson, 1992). The DEC preparation used here was more efficient than the duct homogenate at generating mutagens from BOP. Assuming about 1.5 mg protein (5 × 105 DEC) per filter this gives a mutation frequency of about 66 mutants/106 cells/mg protein (aver- aged over the three times) compared with 28 + 4 (this study, data not shown) and 49 + 1 (Lawson

TABLE 4

MUTAGENICITY IN V79 CELLS OF BOP (0.1 mM) ACTI- VATED BY HOMOGENATES OF HAMSTER DUCT TIS- SUE AND HEPATOCYTES

Mutants/lO 6 cells/mg protein a,b

Normal diet High fat diet

Duct tissue 16.4 5:1.6 (39) 37.9 ± 2.1 (41)

Hepatocytes 3.2 5:0.5 (39) 1.6 ± 0.1 (65)

a Mean+ SE (n = 8); b Absolute survival plating efficiencies (%) in parentheses.

TABLE 5

MUTAGENICITY IN V79 CELLS OF BOP (0.1 mM) ACTI- VATED BY DEC GROWN FOR 8 D IN SDM WITH AND WITHOUT DEX AND INS

Medium Mutants/106 cells a,b

SDM 51 + 4 (27) SDM-DEX 24 ± 4 (26) SDM-INS 45 ± 3 (29) SDM-DEX and INS 25 + 8 (25)

" Mean_+SE, n = 8; b Absolute survival plating efficiencies (%) in parentheses.

et al., 1990) when duct homogenates were used. Our data agree with the finding that whole cells generate more mutagens from BOP and related dialkylnitrosamines than homogenates of those cells (Langenbach, 1986).

The present finding that DEC metabolized BOP after even 8 d in vitro is surprising. Primary cultures of epithelial cells often lose their metab- olizing ability after 3-5 d in vitro even when grown in media (Turner and Pitot, 1989) and under conditions (Wright and Paine, 1992) de- signed to prolong cytochrome P-450 activity. Al- though the medium (WE or SDM) did not affect the activating ability of hepatocytes (Table 3) the possibility existed that some component of the medium was contributing to the maintenance of metabolizing ability of DEC. DEX appeared to be a crucial ingredient as there was a 50% reduc- tion in BOP mutagenicity when it was omitted from the SDM (Table 5). It induces cytochrome P-450 I l i a isoforms in rat liver and hepatocytes, specifically that responsible for testosterone-6/3- hydroxylase activity (McMillan et al., 1991). Its inducing activity in hamsters and extrahepatic tissues appears not be be known and may differ significantly from that ability in rat liver. P- 450IIIA isoforms are involved in the hydroxyl- at±on of aromatic rings on compounds like andro- gens (Miranda et al., 1991) and it is difficult to understand why dialkylnitrosamines would be substrates for these isoforms. An alternative hy- pothesis is that an increase in testosterone metabolites induces other isoforms that are re- sponsible for BOP activation. DEX stimulates P-450IA1, IIB1/2 and IIIE activities in vitro, but much less than IIIA (Wortelboer et al., 1991). A

P-450 IIEl-like isoform is implicated in BOP activation in the pancreas (Kazakoff et al., 1992). However its induction by isoniazid did not in- crease the alkylation of liver or pancreas DNA by BOP in hamsters (Lawson, unpublished results) although P-450IIE1 mRNA levels were increased.

MOP was more efficiently activated by hepato- cytes than by DEC. Langenbach et al. (1980) showed that MOP was 2-3 times more mutagenic than BOP, when hepatocytes were the activating system. In a study of the activating ability of pancreas acinar and duct tissue, not cultured cells, in which DNA damage was the end point MOP produced more DNA damage than BOP (Lawson and Nagel, 1988). It is a more potent inducer of PDA than BOP (Pour et al., 1981) and the failure of DEC to better activate MOP ap- pears inconsistent. MOP would be expected to be more efficiently activated by DEC than hepato- cytes. Different P-450 isoforms are probably re- sponsible for BOP and MOP activation and may not be maintained to the same extent in DEC in vitro. The isoforms responsible for BOP and MOP activation are not known. It is our hypothesis for BOP that the metabolism crucial for its carcino- genicity takes place in DEC not in the liver. This may not be the case for MOP. The present data also indicate that DEN is, at best, a weak inducer of PDA. DEN produces a limited amount of ductular hyperplasia in the hamster pancreas (A- manuma et al., 1991) using a treatment which produces PDA after 10 weeks when BOP is the carcinogen (Mizumoto et al., 1989).

The data presented here show that cells iso- lated from the duct epithelium of the rodent (hamster) pancreas can metabolize xenobiotics (dialkylnitrosamines) to species that possess genotoxic activity. This activity is retained for up to 25 d in vitro. This model may assist in the understanding of xenobiotic-induced pancreas cancer and pancreatitis.

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