Analysis of lacI mutations in Big Blue® transgenic mice subjected to parasite-induced inflammation

Mutation Research 484 (2001) 69–76

Analysis of lacI mutations in Big Blue® transgenic mice subjected to parasite-induced inflammation Olga O. Motorna, Holly Martin, Glenda J. Gentile, James M. Gentile∗ Biology Department, Hope College, 41 Graves Place, Van Zoren Hall, Room 249, Holland, MI 49423, USA Received 1 July 2001; received in revised form 25 July 2001; accepted 8 August 2001

Abstract Parasite infections have long been associated with specific types of human cancers. Schistosoma hematobium is an inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of hepatocellular carcinoma. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica-induced inflammation in mice to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. This provides a perspective on the relationship between chronic inflammation and cancer and may be a model for future studies on this complex association. In previous studies using the Big Blue® transgenic mouse assay, we demonstrated an increase in lacI mutations in liver cells harvested from mice harboring F. hepatica flukes when compared to uninfected control animals. In these studies, we report on the types of mutations associated with this parasite infection. The observed mutational spectrum roughly corresponded to the spectrum of spontaneous mutations in liver cells when compared to control (uninfected) animals. However, the spectrum of mutations from parasitized animals showed a significant increase in complex changes and multiple mutations (18.2%) when compared to what would be expected from control animals (2.8%). © 2001 Elsevier Science B.V. All rights reserved. Keywords: lacI mutations; Big Blue® transgenic mice; Parasite infection; Inflammation

1. Introduction Chronic inflammation resulting from infection has long been associated with specific types of neoplasia. The involvement of hepatitis B virus (HBV) in the etiology of hepatocellular cancer is well-documented [1–3], and herpes viruses [4], reoviruses [5], vaccinia virus and the Epstein–Barr virus [6] are frequently cited as cancer risk agents as well. Additionally, the bacterium Helicobacter pylori was identified by WHO/IARC as a gastric carcinogen ∗ Corresponding author. Tel.: +1-616-395-7190/7542/7714; fax: +1-616-395-7923. E-mail address: [email protected] (J.M. Gentile).

[7]. In most of these instances, cell proliferation and concomitant exposure to exogenous chemical carcinogens have been implicated as compounding factors involved with these unique neoplastic process. Another large group of organisms identified as cancer risk factors in humans and animals are some of the parasitic flukes. Schistisomiasis, a fluke-associated disease, has been well documented as a carcinogenic risk factor (for a review see [8,9]). Schistosoma mansoni is associated with splenic follicular lymphoma, colorectal cancer, hepatocellular cancer and choloangiocarcinoma, S. japonicum is correlated with hepatoma and colorectal cancer, and S. intercalatum and S. hematobium are associated with hydronephro-

0027-5107/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 1 ) 0 0 2 5 8 - 5

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sis, ovarian teratoma, and urinary bladder cancer. Of these, the relationship between S. hematobium and bladder cancer is the most important, and this organism has been classified by WHO/IARC as a Class I carcinogen [7]. Several mechanisms have been proposed to account for the schistosome–cancer relationship, with interactions between the inflammatory response, cell proliferation and altered host metabolism as the most frequently identified processes [10,11]. The liver flukes Opisthorchis viverrini and Clonorchis sinensis are also associated with human cancers, specifically liver cancers. Along with S. hematobium and H. pylori, O. viverrini was identified as a Class I carcinogen by WHO/IARC [7]. Inflammatory processes and endogenous nitric oxide each have been implicated as co-carcinogenic factors in O. viverrini-associated cholangiocellular carcinoma [12,13]. There is additional supporting evidence in O. viverrini infections (and also in HPV infections) that the modification of the cytochrome P450 isozyme CYP2A5 in damaged areas of host hepatic tissue also plays key role in the neoplastic processes [2,3,14]. We have recently found in preliminary studies that mice infected with Fasciola hepatica have a marked reduction in glutathione-S-transferase activity (Gentile, unpublished observation). Using the liver fluke F. hepatica, we have continued the investigation of parasite-associated cancer and genetic damage in order to gain a better perspective on possible mechanisms. F. hepatica is related to O. viverrini and these organisms produce comparable pathology in mammalian systems. Although only occasionally associated with human pathology [15], because of its size and rapid maturation in the host, this organism provides an ideal laboratory model to study the effect of trematode infections in mammalian systems. Previous studies indicate that F. hepatica infections can sensitize mammals, including mice, to exposure to exogenous carcinogens [16,17]. In follow-up experiments, we used the lambda/lacI Big Blue® transgenic mouse model to investigate if genetic damage could result in liver tissue from infection by this digenetic trematode. The frequency of mutation of the lacI gene was higher in Big Blue® transgenic mice infected with parasite F. hepatica when compared to control (uninfected) animals [18]. The data reported here provide perspective on the

nature of the mutations induced from the chronic inflammatory condition that results from infection.

2. Materials and methods 2.1. Parasite and host exposure regimes Metacercarial cysts of F. hepatica were provided by Baldwin Enterprises (Monmouth, OR). Six- to eight-week-old male C57BL/6 Big Blue® transgenic mice were used for the studies. During the exposure studies, each of eight mice was each infected with two F. hepatica metacercaria, suspended in filtered pond water, via oral tube intubation. The control (uninfected) group, which was comprised of four animals, received a dose of the same pond water without the metacercaria via the same mechanism. Infections were allowed to develop over a 15 day period, at which point the animals were sacrificed. The presence of an active infection was verified by visual examination of excised livers and verified by an elevation of eosinophil percentages in the infected animal’s blood profile. A level of eosinophils equal to or greater than 10% per total white blood cell count was set as a standard for a positive infection. 2.2. Isolation of genomic DNA Genomic DNA from the livers of infected and control mice were isolated using RecoverEase® DNA Isolation Kits (Strategene, La Jolla, CA) [19]. In parasite-exposed animals, samples of four separate lobes of the liver (left, right, median, and caudate) were harvested and flash frozen at −80◦ C. Other samples (∼100 mg) from each lobe were preserved in 10% buffered formalin. The formalin preserved tissue was embedded in paraffin, sectioned for slides, stained with hematoxylin and used for histological verification of parasite infection. 2.3. Mutant recovery The lacI transgene was recovered from genomic DNA using the Transpack® Packaging Extract for Lambda Transgenic Shuttle Vector Recovery (Stratagene, La Jolla, CA) was employed [20]. Packaged

O.O. Motorna et al. / Mutation Research 484 (2001) 69–76

phage particles were preabsorbed to SCS-8 Escherichia coli (recA1, endA1, mrcA, ∆[mcrBC-hsdRMS-mrr], ∆[argF-lac], U169, φ80dlacZ∆M15, Tn10[tetr ]) bacterial cells (Stratagene, La Jolla, CA) for 15 min at 37◦ C, mixed with molten (48–50◦ C) NZY top agarose (7%) containing X-Gal (1.5 mg/ml 5-bromo-4-chloro-3-indolyl-␤-dgalactopyranoside) and poured onto NZY plates. Following incubation overnight at 37◦ C the plates were examined on a light box, and blue mutant plaques were scored. In all cases, care was taken to segregate mutant plaques arising from mutations in the lacI transgene in liver genomic DNA from sectored plaques, which can develop in E. coli during the replication of the phage. Mutant plaques were verified for true mutants by repeated plating in the presence of X-Gal. Comparison against positive control mutant plaques was made in each experiment. The mutant frequency was calculated by dividing the number of mutant blue plaques by the total number of plaques plated. 2.4. Sequencing and mutant analysis Sequence analysis of the well-isolated, verified mutant phage was conducted by PCR lacI gene amplification using the Stratagene Big Blue® lacI PCR primer set (Stratagene, La Jolla, CA). For mutants that showed no mutations within the amplified region, additional PCR products were created using two primers located at the base pair positions of −235 to −216, and 1248–1227 and sequence analyzed to check for mutations in outlying regions. PCR products were purified using QIAquick® PCR Purification Kit (QIAGEN, Valencia, CA). The ABI PRISM BIGDYE Terminator Cycle sequencing kit (PE BioSystems, Foster City, CA) and a set of forward and reverse primers for the lacI region [21] were used to prepare the purified PCR samples for analysis. The cycle sequencing products were purified using CENTRI-SEP® columns (Princeton Separations, Adelphia, NJ) and analyzed on an ABI PRISM Genetic Analyzer, Model 310. 2.5. Analysis of mutant sequences The forward and reverse strands of each spacer region were aligned with the use of the Autoassembler

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software package (PE BioSystems, Foster City, CA). The consensus sequence of each mutant was compared to the original non-mutated lacI sequence by the NCBI BLAST program, basic local alignment search tool [22]. Also, resulting alterations in the amino acid sequences in the mutant were aligned and compared to the non-mutated protein of the lacI gene. 2.6. Statistical analysis Differences between mutational spectra of the mutants from infected animals versus spontaneous mutational spectra were compared for statistical significance using the Monte Carlo approximation to Fischer’s exact test as described by Piegorsch and Bailer [23]. Resulting P-values of 0.05% or less were considered statistically significant.

3. Results and discussion Analysis of mutations of the well-characterized lacI gene in the Big Blue® transgenic mouse assay allows for effective extrapolation of in vitro data to whole animal effects [24–27]. We chose, the mouse model for the studies reported here, because of previous data from our laboratory that showed specific metabolic enhancement in mice carrying F. hepatica infections. In those experiments, infected animals were capable of metabolizing AFB1 into its mutagenic form, while non-infected animals were refractory to AFB1 metabolism [17], results similar to those found with mice infected with HBV [2] and hamsters infested with Opisthorchis [14] or C. sinensis [28]. We found similar results using mice that had undergone a partial hepatectomy [29]. Mice are one of the least sensitive species to AFB1 -induced liver carcinogenesis. However, our data on altered metabolic processing of AFB1 in animals with parasite-damaged liver, and data reporting enhanced lacI mutations in transgenic mice challenged by promutagens in concert with mechanical damage (partial hepatectomy) [30] or the glutathione-depleting agent phorone [31], led us to question if the presence of the parasite alone could induce mutations in nearby mammalian host tissues. F. hepatica causes significant liver trauma in infected animals [32] and induces an average

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mutation frequency (MF) for parasite-infected animals of 4.28 × 10−5 at lacI when compared to the average MF of 2.1 × 10−5 for uninfected control animals [18]. We generally found that areas of necrotic tissue proximal to sites of parasite infiltration provided poor and unusable genomic DNA. Thus, we concentrated on harvesting tissue from the damaged lobe of liver, but slightly distal to sites of primary damage. While biliary obstruction comparable to that caused by F. hepatica has been demonstrated to lead to bile duct cell proliferation [33], we have found that the highest amount of cellular activity (cell proliferative and enzymatic) is concentrated in perivenous hepatocytes with a slightly increasing periportal/perivenous gradient rather than in areas more proximal to visible parasite-damaged tissue [34]. These observations are consistent with the fact that most of the hepatic cytochrome P450 content of mouse is normally concentrated in these same areas of the liver [35].

A total of 64 mutants from Big Blue® transgenic mice infected with F. hepatica were sequenced and analyzed. Twenty of these failed to show a sequence alteration between position −235 and 1248 of the base pair fragment. The sequence alterations of the remaining mutants (44 after correction for clonal expansion) are detailed in this report. The spectrum obtained from each mutant was compared to the spectrum of spontaneous mutations in lacI of Big Blue® transgenic mice (Table 1). The compiled data on mutational spectrum from our uninfected (control) animals statistically corresponds to the spectrum of spontaneous mutations in liver tissue from historical and previously published data (Table 1). However, the compiled data from infected animals shows some significant exceptions when compared against spontaneous mutation profiles. The single largest class of mutations consists of single-base substitutions (68.2% of all mutations),

Table 1 Mutation spectrum of parasite-induced and spontaneous mutations Class

Total mutations induced by the parasitea ,e

Spontaneous mutations from control animalsa

Spontaneous mutations reportedb

Mutations

Percentage

Mutations

Percentage

Mutations

Percentage

Transitions G:C → A:T A:T → G:C Transversions G:C → T:A G:C → C:G A:T → T:A A:T → C:G Deletions 1 bp (−1 frameshift) >1 bp Insertions 1 bp (+1 frameshift) >1 bp Complex changesc Multiple mutationsd

21 21 0 9 8 0 0 1 6 3 3 0 0 0 2 6

47.7 47.7 0 20.5 18.2 0 0 2.3 13.6 6.8 6.8 0 0 0 4.6 13.6

12 11 1 4 2 0 2 0 2 1 1 1 1 0 0 0

63.2 57.9 5.3 21.1 10.5 0 10.5 0 10.5 5.3 5.3 5.3 5.3 0 0 0

155 138 17 80 52 11 8 9 29 20 9 10 5 5 2 6

54.9 48.9 6 28.3 18.4 3.9 2.8 3.2 10.3 7.1 3.2 3.6 1.8 1.8 0.7 2.1

Total

44

a

19

282

After correction for clonal expansion. b From historical database [48]. c A complex change is defined as a base pair substitution and/or frameshift and/or deletion/insertion occurring at the same site. d A multiple mutation is scored when more than one mutation occurs in the same mutant with the mutations a few to many base pairs apart. e Significantly different from spontaneous at P < 0.05 according to Monte Carlo approximation to Fischer’s exact test as described by Piegorsch and Bailer [23].

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Table 2 Complex changes in F. hepatica-infected animals Experimental animal I.D.

Mutation type

Base pair positon

Nucleotide alteration

Adjacent bases

IBB3L-B6 IBB6L-A3

Transversion/insertion Double substitution

422–423 66–67

G → TT CC → AT

CAG ATG ATG GGT

predominantly involving G:C base pairs (65.9%). About one half of mutations (47.7%) are G:C → A:T transitions, followed by G:C → T:A transversions (18.2%). The remaining events (31.8%) include deletions and additions of one or more base pair(s) as well as other complex mutational events. Three of the recovered mutants (6.8%) revealed the loss of a single base. One of these mutants was a part of a five-base repeat sequence of adenines (135–139), which is a usual site for spontaneous single base pair insertions/deletions [36,37]. Among the 44 mutants, a total of three with deletions of more than one base pair were recovered. Two of the deletions were observed at a single site (537), and involved the loss of 151 (152) base pair sequence. In general, the percent of large deletions in lacI of Big Blue® mice infected with the parasite (6.8%) was significantly larger than the reported percent of large spontaneous deletions (3.2%). No insertions of one or more base pair(s) were observed (unless they were involved in complex changes or multiple mutations) in all

mutants sequenced, compared to 3.6% reported value for spontaneous insertions. Mutations identified as complex mutations and multiple mutations are noted in Tables 2 and 3, respectively. Two mutants (4.6%) were recovered that had complex changes. One base pair was replaced by two base pairs(G → TT at base pair 422–423); and two adjacent (tandem) base pairs were substituted by different pairs (CC → AT at base pair 66–67). The total percent of complex changes was significantly higher than what one would expect from spontaneous mutation alone (0.7%). Six mutants were recovered that carried more than one mutation (13.6%). Six of the mutations involved transversions, with five of the six being C → A and the other a G → C shift. Only one transition was detected (C → T) and all other mutations involved deletions or insertions of a single base, except in one instance where a deletion of seven bases was identified. The percent of multiple mutations was significantly greater than the one reported for spontaneous

Table 3 Multiple mutations in F. hepatica-infected animals Experimental animal I.D.

Mutation type

Base pair position

Nucleotide alteration

Adjacent bases

IBB1L-B8

Transversion Deletion Deletion Deletion Insertion Insertion Transition Transversion Transversion Transversion Deletion Insertion Insertion Insertion Double substitution

49 812 316 824 293 320 792 777 832 92 1063–1068 19 24 60 67–68

C→A C G G G A C→T C→A C→A C→A ATTAATT C T C CG → AC

ATA GAT GGG TGC GGT TCG GGT CGG GAT CAA GTC GAT ATG GCG TGG GCT TAT TCG TCC GCG GCA GTG CAG GGT TGG TGA CAG AGT TGC GTG

IBB1L-A2 IBB2L-C42

IBB5L-AB14 IBBILM-Y1 IBB2LR-C51

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mutation spectrum (2.1%). In addition, it is worthwhile noting that the number of deletions and insertions uncovered in the mutants with multiple mutations, when combined with the data from deletions and insertions alone, show that changes of this nature are very high (when compared to control and historical data sets) and suggest that this category of genetic damage may have some preferential induction when associated with the parasite-induced trauma. Oxidative damage can play a key role in mutagenesis [38,39] and oxidative stress has been demonstrated to play an important role in mutation induction under certain conditions of chronic inflammation. This is particularly true for the p53 gene in humans [40]. It has been demonstrated that the chronic inflammatory disease ulcerative colitis, a condition which produces reactive oxygen and nitrogen species, increases the risk of colon cancer. Hussain et al. [41] recently reported that the frequency of specific p53 mutated alleles in non-tumorous human colon tissue afflicted with this disease was increased for GC → AT transitions at the CpG site of codon 248 and CG → AT transversions at codon 247 when compared to sequences taken from normal adult controls, strongly suggesting a relationship between higher frequency of mutations under conditions of enhanced oxidative stress. Fasciola, however, has evolved mechanisms to suppress the release of toxic oxygen intermediates in host tissues by activated neutrophils [42] and contains significant amounts of cytochrome c peroxidase, which serves as a protective agent against oxidative damage to deoxyribose [43]. Our data with Fasciola infection demonstrate an increased risk for complex hepatic cell mutations rather than mutations stemming from more definable oxygen radical-associated events, suggesting that a complex set of factors are likely involved in the mutation induction process. We believe that oxidative stress still plays a key role in fluke-induced mutation and in the case of fascioliasis may prove interactive with other mechanisms of mutation to induce the increase in complex changes that we have uncovered. One of the processes that may prove important is the production of ethenobases from lipid peroxidation products [44–46]. We are now investigating the likelihood of etheno base adduct formation as one potential mechanism for the mutational events, which we have observed.

Unique mutation spectrum profiles are not, however, unexpected. It has been reported elsewhere that point-mutational activation of the c-Ki-ras proto-oncogene in human hepatocellular carcinoma differs under different etiological conditions [47]. Point mutations at codon 12 of this proto-oncogene were detected in incipient hepatocellular cholangiocarcinoma in individuals with normal livers, but were not observed in cholangiocellular carcinomas associated with viral infections or fluke infestations. On the whole, our observations regarding F. hepatica-induced lacI mutations in transgenic mice, when taken in concert with other available information, suggest that these mutagenic events might not be characteristic of a specific etiological agent per se, but rather imply a result stemming from broader liver cell damage and incipient associated inflammatory response.

Acknowledgements This research supported by NSF-REU Grant 9322220, Howard Hughes Medical Institute Grants 71191-528501 and 71196-528502, and a grant from the Sherman Fairchild Foundation to support undergraduate research.

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