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Cancer Research 67, 1001-1006, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-3882
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Molecular and Cytogenetical Alterations Induced by Environmental Cigarette Smoke in Mice Heterozygous for Fhit

Silvio De Flora1, Francesco D'Agostini1, Alberto Izzotti1, Nicola Zanesi2, Carlo M. Croce2 and Roumen Balansky1

1 Department of Health Sciences, University of Genoa, Genoa, Italy and 2 Comprehensive Cancer Center, Ohio State University, Columbus, Ohio

Requests for reprints: Silvio De Flora, Department of Health Sciences, University of Genoa, via A. Pastore 1, I-16132 Genoa, Italy. Phone: 39-10-353-8500; Fax: 39-10-353-8504; E-mail: sdf{at}unige.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies in humans and animal models provided evidence that the Fhit gene is an early target for cigarette smoke. We compared the induction of a variety of molecular and cytogenetical alterations in B6-129(F1) mice, either wild type or Fhit+/–, after whole-body exposure to environmental cigarette smoke (ECS) for 15 consecutive days. Both mouse genotypes responded to ECS with a loss of Fhit protein in the bronchial epithelium, accompanied by induction of apoptosis and stimulation of cell proliferation. ECS induced formation of bulky DNA adducts in whole lung. In addition, ECS caused cytogenetical damage both in the respiratory tract and at a systemic level, as shown by a significant increase of micronucleus frequency in pulmonary alveolar macrophages, bone marrow polychromatic erythrocytes, and peripheral blood normochromatic erythrocytes of both wild-type and Fhit+/– mice. These results are compared with those generated in other species, strains, and genotypes of rodents exposed to ECS that we investigated previously. Although the loss of Fhit protein in the bronchial epithelium of ECS-exposed B6-129(F1) mice provides further evidence that the Fhit gene is an early molecular target for ECS, heterozygosity for Fhit does not seem to confer an increased susceptibility of mice in terms of the investigated early biomarkers. [Cancer Res 2007;67(3):1001–6]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chromosome 3p is frequently affected by genomic alterations in a variety of malignancies, also including lung cancer (1). Thus, deletions and/or loss of heterozygosity at 3p, often with a break at 3p14, have frequently been reported in lung tumors, especially in smokers (1). This chromosome harbors FRA3B, the most sensitive among the common fragile sites in the human genome, which is located at the chromosomal band 3p14.2 (1, 2). Ten years ago, the Fhit tumor suppressor gene was identified in this region (3). The Fhit gene is altered by deletion or translocation in many types of cancers, including lung, cervical, gastric, breast, kidney, oral cavity, and colorectal cancers (4). Furthermore, loss of heterozygosity at chromosome 3p14 was found to be more frequent in current smokers compared with never smokers and occurred frequently even in histologically normal or minimally altered bronchial epithelium of smokers, which implies that Fhit might be regarded as a candidate molecular target for cigarette smoke (5, 6). A dose-dependent alteration of Fhit methylation was observed in bronchoalveolar lavage cells from cancer-free patients, as related to the number of cigarettes smoked in a lifetime (7). In addition, the Fhit protein was also reported to be reduced or lost in tumors, including lung, cervical, gastric, and esophageal cancers as well as preneoplastic lesions of the lung (8–11).

Recently, we showed that exposure of rats and mice, belonging to various strains and genotypes, to environmental cigarette smoke (ECS) for short periods, up to 30 days, results in a significant and time-related decrease of Fhit gene expression and a loss of Fhit protein in both pulmonary alveolar macrophages and bronchial epithelial cells (12). Thavathiru et al. (13) showed that benzo(a)pyrene [B(a)P] diol epoxide (BPDE), an ultimate metabolite of the cigarette smoke constituent B(a)P, significantly down-regulates the Fhit gene expression. These findings confirm the hypothesis that Fhit might be regarded as a molecular target for cigarette smoke and its constituents even at early stages of cigarette smoke–related carcinogenesis (14).

The Fhit protein is thought to be involved in proliferation and apoptosis of cancer cells (1, 15–17). In addition, the Fhit protein may be also involved in DNA repair, either directly or indirectly (18). A loss of Fhit protein would be expected to increase vulnerability of DNA to genotoxic agents and to render the genome more susceptible to carcinogen-induced alterations. In fact, Fhit+/– cells showed a higher mutation rate in response to DNA-damaging agents (19).

A possible approach to investigate the role of Fhit protein in mutagenesis and carcinogenesis is to use transgenic mice in which one or both alleles of the Fhit gene are deleted. The murine Fhit gene is similar in sequence, location, and fragility to its human homologue, and Fhit+/– and Fhit–/– mice may serve as a convenient model to study in vivo the effects of Fhit gene alterations in chemically induced mutagenesis and carcinogenesis (1). Fhit-deficient mice, either Fhit+/– or Fhit–/–, were established by inactivating one Fhit allele in mouse embryonic stem cells. These mice displayed an elevated frequency of "spontaneous" tumors and chemically induced tumors (20, 21). The Fhit-deficient mouse model has also been used to prevent tumor development by gene transfer (22, 23). By using (C57BL/6J x 129/SvJ)F1 [B6/129(F1)] mice, either wild type (Fhit+/+) or heterozygous for Fhit (Fhit+/–), we showed that Fhit heterozygosity affects susceptibility of mice to "spontaneous" alopecia patchs and B(a)P-induced preneoplastic lesions of the uterus, whereas there was no change in induction of either lung tumors or forestomach tumors (24).

The goal of the present study was to evaluate whether B6/129 F1 mice, either wild type or Fhit+/–, are susceptible to the induction of early molecular and cytogenetical alterations induced by exposure of mice to high doses of ECS. The results obtained show that, irrespective of the Fhit status, these mice undergo a variety of significant alterations after a 15-day exposure to ECS, including loss of Fhit protein and increase of apoptosis and proliferation in bronchial epithelial cells, formation of bulky DNA adducts in the lung mixed cell population, and induction of cytogenetic damage in the respiratory tract, bone marrow, and peripheral blood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Thirty-four B6-129(F1) mice (14 wild type and 20 Fhit+/–) were bred at the Kimmel Cancer Center (Thomas Jefferson University, Philadelphia, PA) and shipped to the University of Genoa. At the start of the experiment, wild-type mice weighed 36.2 ± 1.79 g (means ± SE), and Fhit+/– mice weighed 34.5 ± 0.72 g. The mice were housed in Makrolon cages on sawdust bedding and maintained on standard mouse chow (MIL, Morini, S. Polo d'Enza, Italy) and tap water ad libitum. The temperature of the animal room was 23 ± 2°C, with a relative humidity of 55%, ventilation accounting for 15 air renewal cycles per hour, and 12-h light-dark cycle. The housing and treatment of mice were in accordance with our national and institutional guidelines.

Treatment of mice. Seven wild-type mice and 10 Fhit+/– mice were kept for 15 days in filtered air and served as sham-exposed controls. The remaining mice were exposed to ECS for 15 days. A whole-body exposure was achieved by burning 1R3 Kentucky reference cigarettes (Tobacco Research Institute, University of Kentucky, Lexington, KY), having a declared content of 22.8 mg tar and 1.5 mg nicotine each, in a smoking machine (model TE-10, Teague Enterprises, Davis, CA). Each smoldering cigarette was puffed for 2 s, once every minute, for a total of 8 puffs, at a flow rate of 1.05 L/min to provide a standard puff of 35 mL. The smoking machine was adjusted to burn five cigarettes at one time and to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%), mimicking exposure to high-dose ECS. Exposure to ECS was 6 h/d, divided into two rounds with a 3-h interval, for 15 consecutive days. This accounted for a daily exposure to the ECS generated by 120 cigarettes. The total suspended particulate in the exposure chambers was, on an average, 113 mg/m3, and CO was 580 ppm.

Collection of biological samples. At time 0 and after 5 and 15 days, peripheral blood drops were collected from the lateral tail vein of all mice and smeared on duplicate slides.

After 15 days, all mice were deeply anesthetized with diethyl ether and killed by cervical dislocation. Bronchoalveolar lavage was immediately done by lavaging the lungs of each mouse with three 2-mL aliquots of cold (4°C) 0.15 mol/L NaCl infused via a cannula inserted into the trachea. The cells were washed twice with PBS and then spun in a cytocentrifuge and fixed with methanol.

The left femur of each mouse was removed and dissected, and bone marrow cells were smeared on duplicate slides.

The lungs were removed. The left lung was stored at –80°C for the analysis of bulky DNA adducts, whereas the right lung was fixed immediately in buffered formalin for 24 h and then embedded in paraffin for apoptosis and immunohistochemical analyses.

Bulky DNA adducts. Lipophilic DNA adducts, enriched with butanol, were detected by 32P post-labeling (25) in lung samples from each one of the 34 mice studied. 32P binding to DNA adducts was catalyzed by T4 polynucleotide kinase (Rockland, Gibertsville, PA), using 64 µCi [{gamma}-32P]ATP, having specific activity ≥6,000 Ci/mmol (ICN, Irvine, CA) as 32P donor. DNA adducts were separated by multidirectional TLC on 10 x 8 cm cellulose sheets coated with polyethylenimine (Macherey and Nagel, Düren, Germany), as previously described (25). Radioactivity was measured by using a 32P imager (InstantImager, Packard, Meriden, CT), and the adduct levels were quantified by calculating the ratio of cells per minute detected in DNA adducts and in normal nucleotides. DNA adducts were analyzed in three separate experiments, in which each sample was tested in duplicate. The results, expressed as DNA adducts/108 nucleotides, are the means ± SE of the average values obtained in the mice composing each experimental group.

Immunohistochemical analyses. Fhit and P53 proteins were detected by immunohistochemistry in formalin-fixed, paraffin-embedded bronchial/bronchiolar epithelium samples from each mouse, using the Histomouse-SP kit (Zymed Laboratories, San Francisco, CA).

In particular, Fhit was detected by means of a rabbit anti-Fhit polyclonal antibody, which was kindly supplied by Dr. Kay Huebner (Ohio State University Comprehensive Cancer Center, Columbus, OH) and used at a final dilution of 1:2,000. Due to the difficulty of assessing the percentage of cells positive for Fhit protein, which appears as a diffuse loss of brownish staining of the cell cytoplasm, the results are expressed as percentage of mice within each experimental group displaying an evident loss of Fhit. The appearance of mouse bronchial epithelia, either positive for Fhit or showing extensive loss of this protein, was shown in a previous article (12).

P53 was detected by means of the CM-5 polyclonal antibody (NCL-P53 CM5p, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom), which detects both overexpression and mutation of the P53 gene. The results, expressed as percentage of P53-positive cells, are means ± SE of the data obtained in the mice composing each experimental group.

Proliferating cell nuclear antigen (PCNA) was detected in slides treated with poly-L-lysine (Poly-Prep slides, Sigma Diagnostics, St. Louis, MO) by using the NCL-PCNA kit (Novocastra Laboratories), which is based on an anti-PCNA monoclonal antibody (clone PC10) and employs the avidin-biotinylated horseradish peroxidase complex technology (ABC technique). The results, expressed as percentage of PCNA-positive cells, are means ± SE of the data obtained in the mice composing each experimental group.

Apoptosis. Apoptotic cells were detected by terminal deoxynucleotide transferase–mediated nick end labeling (TUNEL) method in the bronchial/bronchiolar epithelium, using the Tacs XL Blue Label In situ Apoptosis Detection kit (Trevigen, Gaithersburg, MD). The results, expressed as percentage of apoptotic cells, are means ± SE of the data obtained in the mice composing each experimental group.

Cytogenetical analyses. The cytogenetical damage was evaluated in pulmonary alveolar macrophages (PAM) and bone marrow polychromatic erythrocytes of mice killed at the end of the experiment and in normochromatic erythrocytes (NCE) from peripheral blood samples collected at time 0 and after 5 and 15 days, as described previously (26). Briefly, peripheral blood smears were air-dried and stained with May-Grünwald-Giemsa, and 40,000 NCE per mouse were scored for the presence of micronucleated cells. Bone marrow smears were processed in the same way, and 4,000 polychromatic erythrocytes per mouse were scored for the presence of micronucleated cells. Two hundred bone marrow erythrocytes were scored for calculating the polychromatic erythrocytes/NCE ratio. Methanol-fixed slides of bronchoalveolar lavage were stained with a Giemsa 10% solution, and 1,000 to 2,000 PAM per mouse were scored for the presence of micronucleated and binucleated cells.

Statistical analyses. Comparisons between groups were made by {chi}2 test to assess the significance of the results expressed in terms of frequencies and by Student's t test for unpaired data to assess the significance of the results expressed in terms of means ± SE within each experimental group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As summarized in Table 1 , exposure of B6-129(F1) mice to ECS for 15 days resulted in significant alterations of almost all intermediate biomarkers evaluated.


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  		Table 1. Alterations induced in B6-129(F1) mice, either wild type or Fhit+/–, whole body exposed to ECS for 15 days

 
Fhit and P53 proteins in bronchial epithelial cells. All sham-exposed mice displayed a diffuse positivity for Fhit in virtually all cells of the bronchial/bronchiolar epithelium, whereas exposure to ECS resulted in an evident loss of Fhit in approximately half of the mice, both wild type and Fhit+/–. In contrast, the baseline levels of mutated or inactivated P53, as assessed by using the CM-5 polyclonal antibody, were very low and unaffected by exposure of mice to ECS, irrespective of the Fhit status.

Apoptosis and proliferation in bronchial epithelial cells. The frequencies of apoptotic cells and PCNA-positive cells were very similar in the bronchial epithelium of sham-exposed wild-type and Fhit+/– mice. Irrespective of the Fhit status, exposure to ECS resulted in a statistically significant induction of apoptosis and proliferation. In particular, the proportion of apoptotic cells was increased 2.5x in wild-type mice and 1.8x in Fhit+/– mice, and the proportion of PCNA-positive cells was increased 1.4x and 1.3x, respectively.

Bulky DNA adducts in lung. No well-distinguishable spot was detected in the lung of sham-exposed mice, whereas the mice responded to ECS by increasing the levels of bulky DNA adducts, which were detectable by 32P post-labeling in the form of a typical diagonal radioactive zone containing one unresolved spot in the central area (data not shown). The ECS-related increase of DNA adducts was even higher in wild-type mice (9.6x) than in Fhit+/– mice (5.7x), a difference that was statistically significant.

Cytogenetic analyses in the respiratory tract, bone marrow, and peripheral blood. After 15 days of exposure to ECS, there was a significant increase in the frequency of micronucleated PAM, which was of the same order of magnitude in wild-type mice (3.2x) and Fhit+/– mice (2.5x). Conversely, the frequency of binucleated PAM was not affected by exposure to ECS.

At the same time, there was a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow, which was identical (1.5x) in wild-type and Fhit+/– mice. Irrespective of the Fhit status, the polychromatic erythrocytes/NCE ratio tended to increase in ECS-exposed mice, but the difference with sham-exposed mice was not statistically significant.

The frequency of micronucleated NCE in peripheral blood was similar in the four experimental groups at time 0 and after 5 days, whereas after 15 days, there was a significant increase in ECS-exposed mice, either wild type (2.0x) or Fhit+/– (1.8x).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ECS is a mixture of sidestream cigarette smoke and, in lower proportion, of mainstream cigarette smoke exhaled by active smokers, which can be inhaled by involuntary or passive smokers. ECS is classified as a human carcinogen by the U.S. Environmental Protection Agency (27) and by the IARC (28). Like the majority of known or suspected carcinogens, ECS needs to be assayed at very high doses to yield detectable effects in animal models (29).

The data generated in the present study by using the B6-129(F1) agouti mice support the conclusion that rodents are quite susceptible to induction of ECS-related biomarkers. By disregarding all our previous data obtained with mainstream cigarette smoke, Table 2 shows at a glance the herein reported results compared with the findings of previous studies done in our laboratory by exposing rodents belonging to various species, strains, and genotypes. In all these studies, mice and rats were exposed whole-body to ECS for up to four consecutive weeks, under experimental conditions similar to those described in the present article. The evaluated end points included biomarkers of biologically effective dose, such as (a) adducts to hemoglobin of typical cigarette smoke components (i.e., 4-aminobiphenyl and BPDE; ref. 25) and (b) bulky DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, whole lung, skin bladder, heart, aorta, and liver, both in adults and fetuses (refs. 25, 30–35 and this study); indicators of oxidative stress, such as (c) thiobarbituric acid–reactive substances, resulting from lipid peroxidation, in lung and skin (35) and (d) 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), reflecting oxidative DNA damage in adult lung and skin as well as in fetus liver (25, 30, 31, 35); (e) cell proliferation, evaluated by measuring PCNA in PAM, bronchial epithelium, and skin (refs. 30, 35, 36 and this study); (f) cell apoptosis, evaluated by TUNEL method and by analyzing the expression of apoptosis-related genes in PAM, bronchial epithelium, and skin (refs. 30, 35–37 and this study); (g) expression of multiple genes, as evaluated by analyzing up to 4,858 genes, by cDNA microarrays, in adult lung and liver as well as in fetus liver (31, 33, 38, 39); (h) proteome profiles, as evaluated by analyzing 518 proteins in lung by antibody microarrays (40); (i) specific genes or oncoproteins, such as expression of K-Ras in the lung of mice carrying a mutant P53 transgene (41), decrease of Fhit expression and loss of Fhit protein in PAM and bronchial epithelium (ref. 12 and this study), and P53 mutation or P53 inactivation in bronchial epithelium and skin (refs. 25, 30, 35 and this study); (j) cytogenetical alterations in various cells, including PAM (refs. 25, 30, 35, 42 and this study), polychromatic erythrocytes either in adult bone marrow (refs. 25, 30, 35, 42 and this study) or fetus liver (31), and peripheral blood NCE, in which the time course of micronucleus frequency could be assessed (refs. 25, 30, 35 and this study). All the abovementioned end points were consistently altered by ECS, excepting bulky DNA adducts in adult rat liver (32); proliferation in mouse skin (35); apoptosis in the bronchial epithelium of P53 mutant mice (30); P53 mutation or P53 inactivation in mouse skin (35) and bronchial epithelium (ref. 30 and this study); micronucleated PAM in A/J mice and wild-type (UL53 x A/J)F1 mice (30); polynucleated PAM in A/J mice, (UL53 x A/J)F1 mice, irrespective of the P53 status (30); and B6-129(F1) mice, irrespective of the Fhit status (this study); and micronucleated polychromatic erythrocytes in bone marrow of (UL53 x A/J)F1 mice, irrespective of the P53 status (30).


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  		Table 2. Intermediate biomarkers that we evaluated in various species, strains, and genotypes of rodents, whole body exposed to ECS

 
Thus, the present study provided further evidence that the whole-body exposure of rodents to ECS results in a broad variety of molecular and cytogenetical alterations not only in the respiratory tract but also at a systemic level. This evidence contrasts with the difficulty of inducing lung tumors, whose yield is only weakly enhanced in A/J and other mouse strains exposed to ECS under peculiar experimental conditions (30, 43, 44). We are now trying to overcome this problem by exposing mice within 12 h after birth. The rationale for this approach is that during the perinatal period, there is a tremendous stress, documented by a sharp increase of the levels of both bulky DNA adducts and 8-oxo-dG, accompanied by overexpression of many genes in the lung of newborn mice (45). Preliminary results generated by starting exposure of mice to mainstream cigarette smoke at birth are quite encouraging.3

Heterozygosity for Fhit did not render B6-129(F1) mice more susceptible to the ECS-related molecular alterations investigated in the present study. In fact, there was no appreciable difference between wild-type mice and Fhit+/– mice with respect to the loss of Fhit protein, induction of apoptosis, and stimulation of proliferation in bronchial epithelial cells and to increase of the micronucleus frequency in either PAM, bone marrow, polychromatic erythrocytes, or peripheral blood NCE. The levels of bulky DNA adducts in lung were surprisingly higher in wild-type mice than in Fhit+/– mice, a finding that is of difficult interpretation. DNA adducts are promutagenic lesions that are involved in the initiation of cancer and presumably of other chronic degenerative diseases as well (46). Their levels are the result of a dynamic equilibrium between metabolic activation and detoxification pathways, binding of electrophiles with DNA nucleophilic sites, dilution of molecular lesions due to cell proliferation, and adduct removal via DNA repair mechanisms (46). It has been shown that a preferential repair of BPDE-DNA adducts occurs in the actively transcribed strand (47), which may imply a differential transcription-coupled repair of DNA adducts in wild-type mice and Fhit heterozygous mice. In any case, the difference in ECS-related DNA adduct levels between wild-type mice and Fhit+/– mice does not seem to be biologically relevant because they were well higher in ECS-exposed mice than in sham-exposed mice. The observed patterns may also reflect a different kinetic of DNA adduct formation because, at least in rats, the plateau of DNA adduct formation in lung and other organs is not yet reached after 15 days of exposure to ECS (32). In the present study, we had to choose this exposure time as a compromise for evaluating all monitored end points.

In previous studies done in our laboratories, heterozygosity for Fhit was found to affect susceptibility of B6-129(F1) mice to spontaneous alopecia and B(a)P-induced preneoplastic lesions of the uterus. However, wild-type mice and Fhit+/– mice were equally susceptible to induction by B(a)P of micronucleated NCE in peripheral blood and yield of forestomach tumors. Induction of lung tumors by B(a)P was weak in both genotypes (24). Fhit+/– mice were more susceptible than their wild-type counterparts to the induction of forestomach tumors by N-nitrosomethylbenzylamine (NMBA; ref. 21), whereas there was no difference in spontaneous lung tumors in Fhit heterozygous mice (21). However, the finding that the bulky DNA adducts in lungs were lower in Fhit+/– mice than in wild-type animals seems to be consistent with a previous experiment in which 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK)–induced lung tumors in Fhit heterozygous mice showed an incidence slightly lower than the wild-type counterpart (48). Although in that study DNA adducts were not investigated, NNK, a tobacco-specific lung carcinogen, is known to function by initiating a cascade of reactions that results in production of the adduct O6-methylguanine, among other DNA adducts (49). A strong correlation between persistent O6-methylguanine levels in pulmonary DNA and lung tumors has been observed in mice (50). At this point, it is probably worth studying more in detail the relationship among Fhit protein, DNA adducts of different types, and lung tumor incidence in future investigations. On the other hand, the lung tumor yield in Fhit-deficient mice was increased by Vhl haploinsufficiency, which highlights the role of 3p tumor suppressor combinations in affecting the lung tumor incidence and multiplicity (48).

In conclusion, B6-129(F1) mice seem to be highly susceptible to induction of a variety of molecular and cytogenetical alterations by ECS, alike other previously investigated rodent strains. Although the observed loss of Fhit protein in the bronchial epithelium of ECS-exposed mice provides further evidence that the Fhit gene is an early target for cigarette smoke, Fhit+/– mice were not more sensitive that their wild-type counterparts in developing ECS-related alterations.


   Acknowledgments
 
Grant support: Italian Ministry of Health, Bulgarian Ministry of Education and Sciences, NIH National Cancer Institute, and Sidney Kimmel Foundation for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Current address for R. Balansky: National Centre of Oncology, Sofia 1756, Bulgaria.

3 R. Balansky et al., unpublished data. Back

Received 10/19/06. Accepted 11/29/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. Balansky, G. Ganchev, M. Iltcheva, V. E. Steele, F. D'Agostini, and S. De Flora
Potent carcinogenicity of cigarette smoke in mice exposed early in life
Carcinogenesis, October 1, 2007; 28(10): 2236 - 2243.
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