ELSEVIER Mutation Research 325 (1994) 125-128 Mutation Research Letters

Mutagenicity of heterocyclic amines when activated by pancreas tissue

Terence Lawson *, Carol Kolar Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 S42nd Street, Omaha,

NE 68198-6805, USA

Received 1 May 1994; revision received 12 August 1994; accepted 31 August 1994

Abstract

The heterocyclic amines (HA) 2-aminodipyrido[1,2-a :3',2-d]imidazole (GIu-P-2), 2-amino-3,4-dimethylimidazo- [4,5-f]quinoline (MelQ) and 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) were mutagenic in V79 cells (Chinese hamster lung fibroblasts) using 6-thioguanine resistance as the marker of mutagenicity. Pancreas duct epithelial cells (DEC) from untreated hamsters, homogenates of pancreas ducts from untreated hamsters and those fed a high fat diet and human DEC were used to activate the heterocyclic amines. When hamster cells and tissues were used the optimum mutation frequencies (mutants/106 survivors) measured were: GIu-P-2, 10 + 1; MelQ, 28 + 2 (DEC), 12 + 2 (control, duct homogenate), and 21 + 2 (high fat diet fed, duct homogenate); PhlP, 61 + 5. When human DEC were used the optimum mutation frequencies were: MelQ, 32 + 4; PhIP, 35 + 3.3,8-Dimethylim- idazo[4,5-f]quinoxaline, 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-l-methyl-5H-pyrido[4,3-b]indole were not mutagenic in this assay.

Keywords: Mutagenicity; Heterocyclic amines; V79 cells; Pancreas duct epithelium; Hamster; Human

1. Introduction

Heterocyclic amines (HA), formed when food is cooked and tobacco products are combusted, are carcinogenic in rodents and mutagenic in

* Corresponding author. Tel. (402) 559 7475; Fax (402) 559 4651; Internet TALAWSON@UNMC.EDU.

Abbreviations: BOP, N-nitrosobis(2-oxopropyl)amine; DEC, duct epithelial cells; Giu-P-2, 2-aminodipyrido[1,2- a:3',2-d]imidazole; HA, heterocyelic amines; MelQ, 3,4-di- methylimidazo[4,5-f]quinoline; MelQx, 3,8-dimethylimid- azo[4,5-f]quinoxaline; PhlP, 2-amino-l-methyl-6-phenylimid- azo[4,5-b]pyridine; Trp-P-1, 3-amino-l,4-dimethyl-5H-pyrido- [4,3-b]indole; Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3-b]- indole.

bacteria and V79 cells (Chinese hamster lung fibroblasts) (Eisenbrand and Tang, 1993). There is epidemiological evidence for their involvement in the etiology of human pancreatic ductular ade- nocarcinoma (Anderson et al., 1992) as consump- tion of high protein diets (Bueno de Mesquita et al., 1992) and of tobacco products (Ghadirian et al., 1992) is implicated in this type of cancer. Both are sources of HA.

Pancreatic duct epithelial cells (DEC), the cells of origin of pancreatic adenocarcinoma (Lawson and Kolar, 1992), and duct homogenates (Lawson et al., 1990) activate carcinogenic nitrosamines to mutagenic species. Several nitrosamines with di- verse structures are activated by hamster DEC

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126 T. Lawson, C. Kolar / Mutation Research 325 (1994) 125-128

suggesting that several cytochrome P-450 iso- forms are maintained in vitro. There was the expectation that DEC could activate xenobiotics other than nitrosamines, such as HA. The pres- ent study measures the ability of hamster and human DEC and hamster duct homogenates to activate HA to species that are mutagenic in V79 ceils. The HA examined were 2-aminodipyrido- [1,2-a : 3',2-d]imidazole (GIu-P-2), 3,4-dimethyl- imidazo[4,5-f]quinoline (MelQ), 3,8-dimethyl- imidazo[4,5-f]quinoxaline (MelQx), 2-amino-1- methyl-6-phenylimidazo[4,5-b ]pyridine (PhlP), 3-amino-l,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P- 1) and 3-amino-l-methyl-5H-pyrido[4,3-b]- indole (Trp-P-2).

2. Materials and methods

Animals Male, 8 week old, Syrian hamsters (Unei[SYR])

were used. Some were fed a high fat diet (Don- nelly et al., 1989) for 7 days. The control and high fat diets contained 3.8 kcal/g and 4.8 kcal/g and derived 11% and 46% of their calories from fat.

Research Chemicals, Inc., Downsview, Ont., Canada) were dissolved in DMSO. The concen- tration of DMSO in the medium did not exceed 0.1% (v/v).

Mutagenicity V79 cells do not possess the enzymes (Mc-

Gregor et al., 1992) to activate HA (Lawson et al., 1991). To measure HA mutagenicity in this assay the V79 cells (target cells) were co-cultured with hamster DEC (Lawson and Kolar, 1992), human DEC or with duct homogenates (Lawson et al., 1990) in DME/F12 medium supplemented with NuSerum IV (10%; v/v) (Lawson and Kolar, 1992). Contact was made between the V79 cells and DEC, which were supported on filters, by inverting the filters on top of the V79 monolayer. All co-cultures were for 16 h when the DEC were removed and the V79 cells processed for the mutagenicity assay. Homogenates were removed by washing. Mutagenicity was measured as resis- tance to 6-thioguanine (van Zeeland and Simons, 1976). In each assay three plates were established for determining the plating efficiency and eight plates for the mutation frequency.

Cells Hamster DEC were isolated and cultured

(Lawson and Kolar, 1992). Pancreas duct tissue homogenates were prepared by the method of Lawson et al. (1990). Human DEC were isolated from the perfusate obtained when pancreases were digested for islet cell isolation (Ricordi et al., 1988). DEC were obtained from the cellular material that centrifuged to the bottom of the Ficoll gradient (1.058 g /cm 3 to 1.074 g /cm 3) used to purify the islets. This material was washed three times with HBSS and collected by centrifu- gation. The pellet was applied to a 120/.Lm Bellco stainless steel filter and washed with several vol- umes of HBSS. The material retained on the filter was cultured (Lawson and Kolar, 1992). Hamster hepatocytes were isolated by the method of Williams et al. (1980).

Chemicals GIu-P-2 hydrochloride, MelQ, MelQx, PhlP,

Trp-P-1 acetate and Trp-P-2 acetate (Toronto

3. Results

Irtability of human DEC The age of the donor affected the viability of

the isolated DEC. DEC used in this study were from two male caucasian donors, 18 and 35 years old. Comparable preparations from donors older than 50 years did not produce viable DEC. The smoking habits of the donors are not known. It is assumed that their health status was adequate as they were donors for the UNMC Human Islet Cell Transplant program.

Mutagenicity ,~f HA activated by hamster DEC Glu-P-2, MelQ, MelQx, PhlP, Trp-P-1 and

Trp-P-2 (0-50/zM) were tested in the V79 muta- genicity assay using hamster DEC as the activat- ing system. MeIQx, Trp-P-1 and Trp-P-2 were not mutagenic in this assay. The mutation fre- quencies (mutants/106 survivors) for Glu-P-2, MeIQ and PhIP are shown in Table 1. At the

T. Lawson, C. Kolar / Mutation Research 325 (1994) 125-128 127

Table 1 Mutagenicity of GIu-P-2, MelQ and PhlP after activation by hamster and human DEC

Mutants/106 survivors a

5~M 10~M ~ M 50~M

Glu-P-2 Hamster NT b 10 + 1 0 + 0 NT MeIQ Hamster 9+3 28+2 12-1-3 5-1-1

Human NT 325:4 3:1:1 NT PhIP Hamster 7 + 1 61:1:5 21 + 3 Toxic

Human NT 35 _+ 3 NT NT

a Mean + SE (n = 8), background mutation frequency = 2+ l mutants/106 survivors. b Not tested.

dose which produced the most mutants (10 /xM) they produced mutat ion frequencies of 10 + 1 (GIu-P-2), 28 + 2 (MelQ) and 61 + 5 (PhlP). The data shown in Table 1 are from at least two experiments.

stimuli provided by the high fat diet, e.g. en- hancement of cytochrome P-450 activity. It is not known if the effects of the high fat diet persist in vitro. To ensure the maximum effect of the high fat diet, homogenates were used. When hamster D E C were used the mutagens were active in the following order: PhIP (12.2 x 103 mutan ts /106 survivors/ /zmol) > M e I Q (5.6) > BOP (2.3) > Glu-P-2 (2.0). When human D E C were used the mutagens were active in the following order: PhIP (7.0 x 10 3 mutan ts /106 survivors//~mol) > M e I Q (6.4) > BOP (0.6). When pancreas duct ho- mogenates were used M e I Q produced 4.2 x 103 mutan ts /106 survivors/ /zmol (high fat diet fed hamsters) and 2.4 x 103 mutan ts /106 survivors/ /~mol (control diet fed hamsters).

4. Discussion

Mutagenicity of HA activated by human DEC M e I Q and PhIP were tested in the V79 assay

using human D E C as the activating system. Their mutat ion frequencies (Table 1) were 3 2 _ 4 (MeIQ) and 35 + 3 (PhIP), both at 10/.~M.

Comparison of mutagenicity at doses which gave maximal mutagenicity

The mutagenicity (mutants /106 survivors / /~mol mutagen) of Glu-P-2, MeIQ, PhIP and N- nitrosobis(2-oxopropyl)amine (BOP), a pancreas carcinogen in hamsters (Lawson et al., 1991), activated by hamster and human D E C and ham- ster hepatocytes, is shown in Table 2. The BOP data were obtained in an earlier study (Lawson and Kolar, 1992) and are presented here for comparison. These values are calculated from the mutagenicity at the dose that produced the most mutants and are expressed in this manner to reflect the different doses. For Glu-P-2, M e I Q and PhIP this was 10 tzM, 100/~M for BOP and 25 ~ M for M e I Q when activated by hepatocytes. Da ta for M e I Q activated by pancreas duct ho- mogenates from hamsters fed a high fat diet are also shown. Homogenates were used in these experiments as D E C take about 8 days to reach a sufficient number for use in this assay. During this t ime the D E C would not be exposed to any

Representat ives of three classes of heterocyclic amine, pyridoimidazoles (Glu-P-2), quinolines (MeIQ) and pyridines (PhIP), were metabolized (activated) to mutagenic species by hamster and human pancreas DEC. D E C converted these chemicals to mutagenic species much more effi- ciently than hepatocytes. M e I Q and PhIP were also more mutagenic than BOP, a pancreas car- cinogen in hamsters (Lawson et al., 1991). An involvement of heterocyclic amines in the etiology of human pancreatic cancer has been suggested

Table 2 Comparison of the mutagenicity of BOP, Glu-P-2, MeIQ and PhIP, at the most effective dose

Mutagen Mutants/106 survivors/~mol

Hamster Human

BOP 2.3 x 10 3 0 . 6 × 10 3

GIu-P-2 2.0 × 10 3 NT d MelQ 5.6 x 103 6.4 x 103 MelQ a 0.7 X 10 3 NT MeIQ b 2.4 × 103 NT MeIQ c 4.2x 10 3 NT PhlP 12.2x 10 3 7 . 0 × 10 3

a Hamster hepatocytes; b duct homogenate from hamsters fed a control diet; c duct homogenate from hamsters fed a high fat diet.

Not tested.

128 T. Lawson, C. Kolar /Mutation Research 325 (1994) 125-128

as two lifestyles that are implicated in its induc- tion are the consumption of high protein diets and tobacco products, both of which would be expected to contain HA. There appear to be four studies in the literature in which HA action in the pancreas is reported (Takayama et al., 1989; Lin et al., 1992; Minchin et al., 1993; Turteltaub et al., 1993). These studies report the binding of PhIP metabolites to DNA. In all four the level of binding to pancreas DNA was higher than in any other tissue, including tissues in which PhIP is carcinogenic. This appears to be contrary to the behavior of other HA. As the carcinogenicity of several HA correlates with the extent of DNA binding there is reason to suspect that the pan- creas could be a target for PhIP.

The purpose of the present study was to deter- mine if HA were genotoxic in the pancreas. The data presented here show that pancreas DEC can activate HA to genotoxic species and lend some credence to the idea that HA may be involved in human PDA. We have shown that DEC metabo- lize PhIP much more efficiently than other HA and much more efficiently than hepatocytes sug- gesting, although far from proving, that there would be greater binding to pancreas DNA.

Acknowledgements

This work was supported by grant 90-All from the American Institute for Cancer Research, NCI Cancer Laboratory Core grant CA 36727 and ACS Institutional grant SIG 16. UNMC IACUC (No. 94-017-12) and IRB (No. 119-93-EX) ap- proval were obtained before these studies were initiated.

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Communicated by J.S. Feiton