N-Ethyl-N-nitrosourea-induced transplacental lung tumor development and its control: molecular modulations for tumor susceptibility in a mouse model

Abstract

The development of lung tumors after transplacental N-ethyl-N-nitrosourea (ENU) exposure has been demonstrated in Swiss and Balb/c mice F1 mice. We also suggest the molecular changes that could be affecting the susceptibility to chemical carcinogens during tumor development in the fetus. In this study, ENU administered on the 17^th day of gestation resulted in the formation of lung tumors in Balb/c F1 mice, while only lymphocytic infiltration was observed in Swiss F1 mice, at the end of three months. Molecular changes were observed in both strains, but the degree of alteration in some genes was greater in Balb/c F1 mice than in Swiss F1 mice. Administration of 2% inositol hexaphosphate (IP6) to F1 mice attenuated proliferation, inflammation and upregulated the apoptotic machinery, in terms of the expression of cyclin D1, NF-κβ p50, COX-2, mut p53, Bax, Bcl-2, and Bcl-_XL including caspase enzyme activities and DNA damage. These could be important pathways involved in lung tumor development in offspring of the carcinogen-predisposed mothers; IP6 attenuated lymphocytic infiltration and tumor development by modulating these events. Analysis of the molecular changes and the chemopreventive potential of IP6 during ENU-induced transplacental lung tumorigenesis suggests that the susceptibility to the induction of lung tumors in Balb/c F1 mice could possibly be due to greater over-expression in Balb/c F1 mice than Swiss F1 mice, of genes involved in proliferation and inflammation in transplacental lung tumor development.

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Introduction

Several studies have shown that developing organisms are very sensitive to chemical and physical carcinogens, suggesting that the exposure of pregnant women to environmental toxicants may place the embryo and fetus at a higher risk of the development of cancer. Additionally, different strains of mice exhibit differential susceptibilities to both spontaneous and chemically induced transplacental lung tumorigenesis, due to the interactions between genetic factors and environmental agents that affect the phases of tumorigenesis.¹ Epidemiological and laboratory animal model studies have demonstrated that smoking and exposure to environmental carcinogens by pregnant women could result in the development of cancer in their offspring.² The development of childhood tumors due to exposure to mothers is alarming, and there is a need to explore and understand the basis of transplacental tumor development in order to manage it.³

ENU is a mutagen belonging to the family of N-nitroso compounds (NOCs) that are present in tobacco smoke, fumigants and biohazards, which may affect human health.⁴ Application of ENU has been used in experimental models during the gestation period to study transplacental lung carcinogenesis.^5,6 The testing of mice with ENU allows the estimation of the risks or effects associated with mutagens and carcinogens in the human population.⁷

The outcome of a study using a mouse model could be extrapolated to a situation in humans, as certain mutations described in mouse lung tumors and the histology are similar to those of human lung cancer.^8,9

Over-expression of cyclin D1, a regulator of cell cycle progression from G1 to S phase, has been linked to the development and progression of lung and other cancers.¹⁰ Upregulation of the transcription factor Nf-κβ, a dimer of p50 and RelA (p65), enhances cell survival by transcribing cyclin D1, and is considered a molecular link between chronic inflammation and tumor development¹¹ as it is activated in many types of cancer.^12,13

Over-expression of cyclooxygenase-2 (COX-2) has been seen in approximately 70% of lung adenocarcinomas and is associated with cell proliferation, chronic inflammation and resistance to apoptosis.^14,15 p53, the tumor suppressor gene, is an important defense against cancer as it suppresses tumor growth through cell cycle regulation and also via apoptosis.^16–18 Mutations in the p53 gene facilitate the development of resistance to apoptosis and increase the survival of the damaged cells. Upregulation of Bax and downregulation of Bcl-_XL and Bcl-2 lead to the activation of the key implementers of apoptosis, caspase-3 and caspase-9,^19,20 which have been well documented in a p53-dependent pathway.²¹

There is an immense need to develop new therapeutic strategies to ameliorate lung cancer for better management. In order for the molecular modulations which occur during tumor development to be of use as targets for therapy, they need to be responsive to antitumor agents. Inositol hexaphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, is present in foods like rice, grains, fruits and vegetables. A substantial amount of IP6 (0.01–1 mM) is present in mammalian cells and it exerts its effect after being converted into lower phosphates.^22–25 It is reported that IP6 can inhibit many types of cancer in different experimental models, including prostate, skin, mammary, colon, lung and liver.^26–30 IP6 has been shown to reduce tumor volume, tumor weight, cell proliferation, angiogenesis and induce apoptosis^25,31,32 but its molecular mechanisms needs to be explored.

Here we demonstrate ENU-induced transplacental lung tumorigenesis in F1 mice, in the presence or absence of IP6, and study its underlying basis with an emphasis on the representatives of the critical pathways involved in tumorigenesis. This is achieved by assessing cyclin D1, Nf-κβp50, COX-2 and mut p53, and the balance between pro-apoptotic events such as Bax, caspase-3, caspase-9, and anti-apoptotic events such as Bcl-2, Bcl-_XL, in mouse lungs. Chemopreventive agents could inhibit Nf-κβ activation and block the Nf-κβ signaling cascade.³³ Our results would help in the better understanding of the mechanisms of transplacental tumor development, the management of the disease by IP6 with a novel molecular basis, and the role of IP6 in lung tumor development and the susceptibility for it in the progeny of exposed mothers.

Material and methods

Animals

Female Swiss and Balb/c mice (6–8 weeks old and with 18–22 g body weight) from the inbred colony of CSIR-IITR, Lucknow, were used throughout the study. Male Swiss and Balb/c mice were used only for mating. All the animals were kept in a 12 h light and dark cycle, and given a pellet diet and water ad libitum. This pellet diet, which consisted of 21.53% protein; 5.24% fat, 5.3% crude fiber; 57.59% carbohydrate and 363.64 kcal per 100 g caloric value, was procured from M/S Ashirwad Pvt. Ltd, Chandigarh, India.

The study was approved by, and the animals were handled according to the norms of, the Institutional Animal Ethics Committee (IAEC).

Chemicals

N-Ethyl-N-nitrosourea and inositol hexaphosphate were procured from Sigma Chemical Co. USA. AMV-RT-PCR kits, Taq DNA polymerase, dNTPs, PCR primers and bovine serum albumin (BSA) were from Bangalore Genei, India. Enzyme activity assay kits for caspase-3 (Cat no. BF3100) and for caspase-9 (Cat no. BF10100) were procured from R&D Systems, USA. Primary rabbit polyclonal COX-2 and Bcl-2 antibodies were purchased from Alexis Biochemicals, USA; rabbit polyclonal Bcl-_XL, Bax, Nf-κβp50 or cyclin D1 and goat polyclonal caspase-3 antibodies were purchased from Santa Cruz Biotechnology, USA; mouse polyclonal mut p53 antibody was purchased from Boehringer Mannheim, Germany and rabbit polyclonal caspase-9 antibody was purchased from Cell Signaling Technology, USA. IgG, HRP-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were obtained from Bangalore Genei, India. The rest of the chemicals were procured from local commercial sources and were of analytical grade.

Chronic animal bioassay

Mating was initiated for both the Swiss and Balb/c strains by placing one male with two female mice for a night. Day zero of pregnancy was determined by checking the presence of a vaginal sperm plug next morning. Thereafter, following impregnation, mice (twelve in total) were housed individually. One group of pregnant mice (six in total) received only 50 mM citrate buffer of pH 6.0, while another set of pregnant mice (six in total) received ENU intraperitoneally (i.p.) at a dose of 40 mg kg⁻¹ body weight in 50 mM citrate buffer of pH 6.0, on the 17^th day of gestation. Pups (litter size 4–8 per dam; body weight 1–1.5 gram per pup) were delivered after the 18^th day of gestation and weaning of the pups was done at the age of 24 days. Untreated pups were further divided into two groups, 1 and 2. Group-1 pups (control) were put on normal drinking water and group-2 pups (IP6) were put on 2% IP6 in drinking water. Pups from the ENU-treated mothers were also divided into two groups, 3 and 4. Group-3 pups (ENU) received normal drinking water and group-4 pups (ENU+IP6) received 2% IP6 in drinking water. Treatment details are given in Table 1. Dose selection of ENU was based on our and others’ previous reports^3,34 and the IP6 dose was also based on earlier reports.^31,32,34 Considering that a mouse drinks a daily average of around 5 ml of water, each animal consumed around 100 mg of IP6 in a day.

Table 1 Experiment design and treatment schedule

Treatment on the 17^th day of gestation	Groups	Number of pups (N)		Weaning (days)	Treatment (after weaning)
		Swiss	Balb/c
50 mM citrate, pH 6.0 (i.p.), once. (no. of pregnant mice = 6)	1	14	15	24	Normal drinking water
	2	14	15	24	2% IP6 in drinking water
ENU (i.p.) 40 mg kg^−1.bwt in 50 mM citrate, pH 6.0, once. (no. of pregnant mice = 6)	3	15	15	24	Normal drinking water
	4	14	18	24	2% IP6 in drinking water

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Swiss and Balb/c F1 mice (body weight 23–30 g per mouse) from each group were sacrificed by cervical dislocation three months after ENU exposure. The lungs were harvested, and tumors on the surface of the lungs were counted and their sizes determined under a dissecting microscope. Lungs from one third of the mice from each group were fixed in 10% phosphate-buffered formalin, and lungs from the rest of the mice were taken out and processed for molecular studies.

Histopathological analysis

Formalin-fixed lung tissues were dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in paraffin to prepare the blocks. All five lobes of the lungs were sectioned, mounted on slides and stained. The 5 micron serial sections were stained with hematoxylin-eosin and were examined using a Leica DFC 295 camera under a Leica DM 1000 microscope, at a magnification of 40× for histopathological evaluation and for counting the tumors. The diameter of the largest section of each tumor was measured using the Leica Live Measurement software and the area of the tumor was calculated in terms of mm².¹¹

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated using TRIzol (Invitrogen, USA) as per the manufacturer's instructions. RNA was treated with DNaseI (Ambion Co. USA) to remove any DNA contamination. cDNA was synthesized by RT-PCR using an AMV-RT kit as per instructions. cDNA (1 μl) was used as a template for amplification using mRNA specific primers for specific genes. Nucleotide sequences (MWG, Biotech, Germany) for the gene specific primers are listed in Table 2. PCR was performed after denaturation at 95 °C for 5 min, for 35 cycles (95 °C for 60 s, annealing for 60 s, and amplification at 72 °C for 60 s), followed by a final extension for 4 min at 72 °C. PCR products were resolved on 1.5% agarose gel and visualized using ethidium bromide. Quantification was done using Gene Tool Synegene software.¹¹

Table 2 Nucleotide sequences of primers and PCR product size for RT-PCR

Gene	Nucleotide sequences	Annealing temperature (°C)	Product size (bp)
COX-2	F 5′-GTGGAAAAACCTCGTCCAGA-3′	60	256
	R 5′-TGATGGTGGCTGTTTTGGTA-3′
Cyclin D1	F 5′TGTTCGTGGCCTCTAAGATGAAG-3′	58	136
	R 5′AGGTTCCACTTGAGCTTGTTCAC-3′
Mut p53	F 5′-ATGACTGCCATGGAGGAGTC-3′	58	663
	R 5′-CTCGGGTGGCTCATAAGGTA-3’
Nf-κβ p50	F 5′GCACAGACGGTGTCTAGCAA-3′	58	131
	R 5′GCGGAGGGACAGCAGTAACA-3′
Bcl-2	F 5′- AGCCCGTGTTTGTAATGGAG-3′	58	476
	R 5′- CACAGCCTTGATTTTGCTGA-3′
Bcl-XL	F 5′-AGGCAGGCGATGAGTTTGAAC-3′	61	399
	R 5′-GAACCACACCAGCCACAGTCA-3′
Bax	F 5′-TGTTTGCTGATGGCAACTTC-3′	58	104
	R 5′-GATCAGCTCGGGCACTTTAG-3′
Caspase-3	F 5′-AGGGGTCATTTATGGGACAAA-3′	57	127
	R 5′-TACACGGGATCTGTTTCTTTG-3′
Caspase-9	F 5′ -CAGGCCCGTGGACATTGGTT-3′	62	438
	R 5′-CAGCCGCTCCCGTTGAAGATA-3′
β-Actin	F 5′-TGTGATGGTGGGAATGGGTCAG-3′	60	514
	R 5′-TTTGATGTCACGCACGATTTCC-3′

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Western blot analysis of proteins

The lung tissue extract was prepared in 20 mM Tris buffer (pH 7.5) consisting of 2 mM sucrose, 2 mM EDTA, 0.5 mM EGTA, 2 mM MgCl₂, 2 mM PMSF, 1 mM DTT, 0.03 mM Na₃VO₄ and protease inhibitor cocktail (Sigma Chemical Co. USA). An aliquot equivalent to 50 μg protein was subjected to SDS PAGE on 10–12.5% Tris-glycine gel. The separated proteins were then transferred onto an Immobilion-P-membrane (PVDF) (Millipore, USA) and were probed with COX-2, Bcl-2, Bcl-_XL, Bax, Nf-κβp50, cyclin D1, caspase-3, mut p53 or caspase-9 antibodies in 5% nonfat milk powder in TBST (100 mM Tris-HCl and 150 mM NaCl and 0.1% Tween-20), at a dilution of 1 : 2000, along with a peroxidase-conjugated appropriate secondary antibody (Bangalore Genei, India). Signals were visualized using a Chemoluminescence HRP detection system (Millipore, USA)¹¹ and quantified on Versa Doc (Bio-Rad). Membranes were stripped for reprobing with an antibody for β-actin (Sigma Co.).

Estimation of enzyme activity of caspase-3 and caspase-9

The lung tissue extract was prepared in the lysis buffer provided in the caspase assay kit. 10 000 g supernatant was used to determine the caspase activity using a colorimetric protease assay kit that included specific substrates for caspases-3 and -9, according to the manufacturer's instructions. Briefly, in a 96-well plate, tissue lysate equivalent to 100 μg protein in 50 μl was added to 50 μl 2× reaction buffer containing 10 mM DTT and 5 μl of the 4 mM substrates DEVD-pNA for caspase-3, or LEHD-pNA for caspase-9 (provided with the kit) followed by incubation at 37 °C for 2 h. At the end, absorbance was measured at 405 nm in a micro plate reader (BMG Labtech, USA).²⁴ Caspase activity was expressed in terms of optical density per mg protein per h in figures.

TUNEL assay

This assay was based on the identification of DNA fragmentation, a hallmark of apoptosis, using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The procedure was performed using a TACS 2TdT DAB kit (Trevigen, USA,) as per the manufacturer's protocol.³⁵ After deparaffinization, samples were rehydrated in decreasing concentrations of ethanol (100%, 90%, and 70%). TUNEL positive signals were visualized with a horseradish peroxidase-mediated diaminobenzidine (DAB) staining solution reaction. Cell nuclei were counterstained with a methyl green solution. Then samples were dehydrated in increasing concentrations of ethanol (70%, 90%, and 100%) followed by DPX mounting.

Protein estimations were done with Bradford reagent (Bio-Rad Laboratories, Inc., USA) using BSA as a standard.

Statistical analysis

Data were expressed as mean values ±SD. Student's ‘t’ test was used to analyze the difference in tumors between the ENU- and ENU + IP6-treated groups. Statistical analysis of gene expression was performed using Graph Pad Prism (version 5.0, Graph Pad Software Inc., USA). ANOVA was performed to compare the means of the groups, and statistical significance was determined in terms of the ‘p’ values. Analysis using the Newman–Keuls method was done to compare significant changes among all the groups.

Results

Transplacental lung tumor development in F1 mice

We evaluated tumor development by gross and microscopic examination at the end of a three month period following a single dose of ENU, administered on the 17^th day of gestation, and found that adult Balb/c F1 mice are more susceptible to tumor induction than Swiss mice.

Gross examination

No tumors were observed on the lung surfaces of Swiss F1 mice. Meanwhile, tumors were clearly observed on the lung surfaces of 12 out of 15 Balb/c F1 mice 3 months after ENU exposure (Fig. 1-I). The total number of visible lung tumors (multiplicity) was 92, in 12 tumor-bearing mice. When 2% IP6 had been administrated, 15 out of 18 mice developed tumors and the total number of tumors was 65. There was a reduction in the average number of tumors, from 7.6 to 4.3, resulting in a 44% inhibition of tumor development following administration of IP6 (Fig. 1-I). There was no effect of ENU on the number of litters, and the body and lung weights of the F1 mice also remained unaffected.

Fig. 1 Evaluation of ENU-induced development of transplacental lung tumors in both Swiss and Balb/c F1 mice at the end of three months. Gross (I) examination of tumors in tumor-bearing mice and microscopic (II, III) examination of the lungs of both Swiss and Balb/c F1 mice. Lymphocytic infiltration in Swiss F1 and tumors in Balb/c F1 are shown by the arrows. Quantitative analysis of tumor status and tumor area in Balb/c F1 mice is shown by histograms (I, III). Bars represent the mean values ±SD. ^†P < 0.05, ^††P < 0.01. Magnification = 40×.

Microscopic examination of lung tumors in wax embedded lung sections

Microscopic examination of the lungs was done in F1 mice from both strains. We did not observe any tumors in Swiss F1 mice from ENU-exposed mothers, but some lymphocytic infiltration was observed in one third of the mice used for the microscopic examination. This lymphocytic infiltration was attenuated by IP6 administration (Fig. 1-II). Microscopic evaluation for the Balb/c strain was done in 5 F1 mice from the ENU-exposed group and 6 F1 mice from the ENU-treated group drinking 2% IP6. At the end of 3 months, clearly visible defined adenomas were observed in 3 Balb/c F1 mice (Fig. 1-III). The total number of tumors in tumor-bearing F1 mice from the ENU-exposed Balb/c mothers was 23, with an average of 7.6 tumors per tumor-bearing mouse. With the administration of 2% IP6, this number was reduced to 10 in 3 tumor-bearing F1 mice, with an average of 3.33 tumors per tumor-bearing F1 mouse; this equated to a 57% reduction in tumor development (p < 0.05) (Fig. 1-III). Judging by the area of the largest section of each tumor, a 60% inhibitory effect of IP6 was observed on the size of the tumors (p < 0.01). Tumor sizes ranged between 0.231–0.685 mm² with an average area of 0.452 mm². Tumor size was reduced to a range between 0.020–0.390 mm², with an average of 0.181 mm² tumor area, in mice receiving 2% IP6 (Fig. 1-III).There were no lung adenomas in the group receiving IP6 alone. Citrate buffer was used as a vehicle and no visible effects were observed in Swiss or Balb/c F1 mice.

Status of proliferation- and inflammation-associated genes

In order to find the genes responsible for tumor susceptibility, we tried to evaluate the transplacental effect of ENU on the expression of critically important representative genes of proliferation and inflammation, in both Swiss and Balb/c F1 mice. Cyclin D1, Nf-κβp50 and COX-2 were found to be upregulated by ENU exposure (Fig. 2). The upregulation in the mRNA of cyclin D1, Nf-κβ p50 and COX-2 was 37%, 24% and 32% in Swiss F1 and 79%, 36% and 40% in Balb/c F1 mice with respect to the control. After administration of IP6, the ENU-induced mRNA increases were 16%, 15% and 14% in Swiss F1 and 55%, 0% and 25% in Balb/c F1, with respect to the control (Fig. 2-I).

Fig. 2 Evaluation of proliferation and inflammation markers in F1 Swiss and Balb/c mice. Analysis of cyclin-D1, NF-κβ p50 and COX-2 mRNA by RT-PCR (I), and protein by western blotting (III), is shown for both Swiss and Balb/c F1 mice. Quantitative analysis is shown by histograms (II, IV). Three individual samples were analyzed for the expression of genes and were subjected to statistical analysis. Bars represent mean values ±SD. *^,†P < 0.05; **^,††P < 0.01. *^,** compared with the control and ^†,††compared with ENU. Swiss, Balb/c.

The increases in the levels of cyclin D1, Nf-κβ p50 and COX-2 proteins were 89%, 56% and 36%, respectively, in Swiss F1 mice. Meanwhile, in Balb/c F1 mice, the increases for the three proteins were 200%, 141% and 40%, respectively, compared to the control. After administration of 2% IP6, the ENU-induced increases were attenuated for the three proteins, but remained 55%, 43% and 20%, respectively, in Swiss F1, and 29%, 84% and 9%, respectively, in Balb/c F1 mice compared to the control (Fig. 2-III).

Modulation of apoptotic genes

Various pro- and anti-apoptotic proteins play crucial roles in programmed cell death. We tried to evaluate how far the status of the pro- or anti-apoptotic proteins p53, Bax, caspase-3, caspase-9, Bcl-_XL and Bcl-2 is responsible for governing susceptibility towards ENU-induced tumor development in Swiss and Balb/c F1 mice in our study.

ENU exposure resulted into an upregulation of mutp53, Bcl-_XL and Bcl-2 in F1 mice (Fig. 3). The upregulation in the mRNA of mutp53, Bcl-_XL and Bcl-2 was 29%, 31% and 31%, respectively, in Swiss F1 mice, while the expressions of these three genes were increased by 38%, 82% and 62%, respectively, in the Balb/c F1 mice as compared to the control. After administration of 2% IP6, upregulation of the mRNA of the three genes was reduced to 8%, 7%, and 8%, respectively, in Swiss F1 and 0%, 59% and 15%, respectively, in Balb/c F1 mice compared to the control (Fig. 3-I). At the protein level, upregulation of mut p53, Bcl-_XL and Bcl-2, was 67%, 44%, and 61%, respectively, in Swiss F1, which increased to 76%, 195% and 126%, respectively, in Balb/c F1 mice from the ENU-exposed mothers as compared to the control. After administration of 2% IP6, the upregulation was reduced to 38%, 6% and 19%, respectively, in Swiss F1 mice and 4%, 25% and 52%, respectively, in Balb/c F1 mice compared to the control (Fig. 3-III).

Fig. 3 Analysis of mut p53, Bcl-_XL and Bcl-2 mRNA by RT-PCR (I) and protein by western blotting (III) are shown for both Swiss and Balb/c F1 mice. Quantitative analysis is shown by the histograms (II, IV). Three individual samples were analyzed for the expression of genes and were subjected to statistical analysis. Bars represent mean values ±SD. *^,†P < 0.05; **^,††P < 0.01. *^,**compared with control and ^†,††compared with ENU. Swiss, Balb/c.

The pro-apoptotic genes Bax, caspase-3 and caspase-9 were downregulated in the F1 generation from ENU-exposed mothers. The ENU-induced decreases in the levels of Bax, caspase-3 and caspase-9 were 34%, 28% and 24%, respectively, at the mRNA level, and 17%, 38% and 27%, respectively, at the protein level, in Swiss F1 mice. Meanwhile, the downregulation of the three genes was 27%, 20% and 47%, respectively, at the mRNA level, and 28%, 42% and 40% at the protein level in Balb/c F1 mice as compared to the control. Administration of IP6 elevated the expression of the three genes by 26%, 4% and 15%, respectively, at the mRNA level, and 8%, 21% and 0% at the protein level in Swiss F1 mice. Meanwhile, the upregulation was 6%, 46% and 11% at the mRNA level and 6%, 14% and 16% at the protein level in Balb/c F1 mice, as compared to the control (Fig. 4-I, III).

Fig. 4 Analysis of Bax, caspase-3 and caspase-9 mRNA by RT-PCR (I) and protein by western blotting (III) is shown for both Swiss and Balb/c F1 mice. Quantitative analysis is shown by the histograms (II, IV). Three individual samples were analyzed for the expression of genes and were subjected to statistical analysis. Bars represent mean values ±SD. *^,†P < 0.05; ^††P < 0.01. *compared with the control and ^†,††compared with ENU. Swiss, Balb/c.

Bax/Bcl-2 ratio at protein and mRNA level

An increase in the Bax : Bcl-2 ratio corresponds with the onset of apoptosis. We observed that ENU exposure resulted in a decrease in Bax : Bcl-2 ratios, both at the mRNA and protein levels. The decreases were 51% and 50% in Swiss F1 mice and 55% and 57% in Balb/c F1 mice for mRNA and protein levels respectively, as compared to the control. Administration of IP6 helped to attenuate this decrease, leading to only 33% and 17% decreases in Swiss F1 and 12% and 28% decreases in Balb/c F1 mice for mRNA and protein levels respectively, as compared to the control (Fig. 5-I, II).

Fig. 5 Assessment of the apoptotic events in Swiss and Balb/c F1 mice. Quantitative analysis of the Bax : Bcl-2 ratios for mRNA and protein levels in both Swiss and Balb/c F1 mice is shown by histograms (I, II). Three individual samples were analyzed for the expression of genes. Quantitative analysis of caspase-3 and caspase-9 enzyme activity is shown by histograms (III, IV). Three individual samples were analyzed in triplicate for the enzyme assay and were subjected to statistical analysis. Bars represent mean values ±SD. *^,†P < 0.05; **^,††P < 0.01. *^,**compared with the control and ^†,††compared with ENU. Evaluation of apoptosis by TUNEL assay in lung tissue sections of both Swiss and Balb/c F1 mice is shown in (V). Fragmented DNA in apoptotic cells was stained brown with DAB and is shown by arrows. Magnification = 100×. Swiss, Balb/c.

Enzyme activity of caspase-3 and caspase-9

Having demonstrated the expression of the caspase-3 and caspase-9 genes, we tried to show their functionality in terms of the enzymatic activity of caspase-3 and caspase-9, in both Swiss and Balb/c F1 mice from ENU-exposed mothers. The enzyme activities of caspase-3 and caspase-9 were inhibited by 26% and 43%, respectively, in Swiss F1 mice and 63% and 67%, respectively, in Balb/c F1 as compared to the control. ENU-induced inhibitions of the activity of the two enzymes after administration of IP6 were reduced and were only 11% and 25%, respectively, in Swiss F1 and 25% and 4%, respectively, in Balb/c F1 as compared to the control (Fig. 5-III, IV).

DNA breakdown as detected by TUNEL assay

In order to show the status of apoptosis, we evaluated the DNA breakdown using a TUNEL assay (Fig. 5-V). There was no DNA fragmentation in either the Swiss or Balb/c F1 mice from ENU-exposed mothers. DNA fragmentation, as indicated by the intensity of the brown stain, appeared to be increased after administration of IP6 (arrow in Fig. 5-V) in both Swiss and Balb/c F1 mice. However, the intensity of the brown stain in the stained cells, which indicated DNA fragmentation and thus the increased incidence of apoptosis following IP6 administration, appeared to be same in both Swiss and Balb/c F1 mice. This suggests that the apoptotic events are altered in F1 mice irrespective of the development of tumors.

Discussion

We hypothesize that the risks associated with in utero exposure to environmental carcinogens could be due to alterations at the molecular level. We tried to demonstrate these molecular alterations with respect to the susceptibility towards the effects of a tumorigen in both Swiss and Balb/c F1 mice, from mothers exposed to ENU on the 17^th day of gestation; this was because the transplacental induction of lung tumors in offspring is affected by the day of gestation on which carcinogen treatment occurs.³ ENU, a direct-acting carcinogen, did not result in tumors on the lung surfaces, but caused distinct histopathological changes in terms of lymphocytic infiltration in Swiss F1 mice. However, over the same time period, well-defined tumors appeared on the lung surfaces and adenomas were observed in Balb/c F1 mice. These alterations were sensitive towards the presence of an antitumor agent, IP6, in both strains. After administration of IP6, lymphocytic infiltration was attenuated in Swiss F1 mice and a significant reduction in the number and size of lung tumors was observed in Balb/c mice. These results are supported by earlier studies where we had demonstrated the appearance and prevention of lymphocytic infiltration and hyperplasia in mouse lungs.³⁴

The differential tumorigenic effects provided the basis for further evaluation of the molecular modulations which might account for the differential susceptibility in these strains,³⁶ as well as the modulatory effects of IP6 towards ENU exposure³⁴ in F1 mice. Cyclin D1 and nuclear factor (Nf-κβ) play a major role in lung tumorigenesis by regulating several pathways involved in tumor development.¹⁰ Nf-κβ, p50 and cyclin D1 were upregulated in both Swiss and Balb/c F1 mice, but Nf-κβ and cyclin D1 appeared to be more responsive in Balb/c F1 mice compared to Swiss F1 mice. COX-2 plays an important role in the inflammatory process and is over-expressed both in human and mouse models. Our results on the levels of COX-2 expression are in agreement with published reports showing over-expression of COX-2 in tumorigenesis. However, we could not observe any difference in COX-2 gene expression in Swiss or Balb/c F1 mice.

Apoptosis is inhibited or slowed down during tumor development and its restoration is mediated by the activation of caspase-3 and caspase-9.³⁷ We also showed the downregulation of both the expression and the functional activities of pro-apoptotic caspase-3 and caspase-9 gene in response to ENU during transplacental lung tumor development in Balb/c F1 mice, and also in Swiss F1 mice where tumors did not develop. This suggests that the apoptotic events are disrupted and could lead to the reduction of apoptosis in predisposed conditions, irrespective of the appearance of tumors.

We substantiated the hypothesis that apoptotic events play an important role in tumorigenic susceptibility by analyzing the Bcl-2 family of proteins. IP6-induced apoptosis was associated with an upregulation of Bax and downregulation of Bcl-2; this led to caspase-3 and caspase-9 activation and DNA fragmentation (TUNEL), resulting in the induction of apoptosis in a p53-dependent pathway.³⁸ The high level of p53 increased the ratio of Bax : Bcl-2 and resulted in the induction of apoptosis.³⁹ Likewise, the inverse effect on Bax and Bcl-2 expression in F1 mice from the ENU-exposed mothers was attenuated after the administration of IP6. Reduction in the Bax : Bcl-2 ratio from the ENU-exposed population appeared to be same in Balb/c or Swiss F1 mice, and similar effects of IP6 were observed in both strains. This suggests the importance of the apoptotic events irrespective of the appearance of tumors, and indicates that tumorigenesis involves other associated molecular modulations.

Since IP6 inhibited tumor development and restored the altered apoptotic events, these results imply that IP6 might exert its chemopreventive effects by upregulating the apoptotic pathways in the F1 generation. IP6-induced apoptosis in terms of the levels of p53, Bcl-_XL, Bax/Bcl-2 ratio, caspase-3, and caspase-9 resulted in the restriction of ENU-induced transplacental lung tumorigenesis.²⁴ In addition, the decrease in expression levels of COX-2, Nf-κβ, p50 and cyclin D1 could also be the basis of the chemopreventive effects of IP6 during transplacental lung tumorigenesis in F1 mice, as suggested previously.^10,11

These results suggest that molecular changes occur even before the onset of tumors. Furthermore, the degree of gene expression may be associated with different susceptibilities to the development of lung tumors in the F1 generation from mothers predisposed to a tumorigen.⁴⁰ The genes involved in cell proliferation, chronic inflammation and apoptosis were more responsive toward the ENU-induced transplacental lung tumorigenesis in Balb/c F1 than in Swiss F1mice. The strain-dependent differences in these altered genes might have subsequently created microenvironments, playing vital roles in transplacental lung tumor development.¹ Therefore, we could say that Balb/c F1 mice are more sensitive towards lung tumor induction, while Swiss mice are less sensitive over the given time period. It also implies that the chemopreventive effects of IP6 appear to be mediated by the regulation of the cyclin D1, Nf-κβ, COX-2, Bcl-2, Bcl-_XL, Bax, caspase-3, caspase-9, and p53 genes in ENU-induced transplacental lung tumorigenesis. Our data would be useful in exploring molecular alterations for differential susceptibility for tumor development, and its control.

Conflict of interests

The authors report no conflicts of interest.

Funding

This work was supported by the Indian Council of Medical Research and University Grants Commission, New Delhi. India.

Acknowledgements

The authors would like to thank the Director of the CSIR-Indian Institute of Toxicology Research, Lucknow for providing the facilities, and Ms Pratima Uppadhyay for her help in histopathological work.

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Footnote

† CSIR- IITR communication no. 3130.

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