Elsevier Editorial System(tm) for Journal of Hazardous Materials Manuscript Draft
Manuscript Number: HAZMAT-D-10-03627
Title: Synergism between 2,3,7,8- tetrachlorodibenzo-p-dioxin and 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone on lung tumor incidence in mice
Article Type: Research Paper
Keywords: Keyword: 2,3,7,8- tetrachlorodibenzo-p-dioxin, P16, lung cancer Corresponding Author: Dr Pinpin Lin, Ph.D.
Corresponding Author's Institution: National Health Research Institutes First Author: Ying-Jan Wang, Ph.D.
Order of Authors: Ying-Jan Wang, Ph.D.; Han Chang, M.D., Ph.D.; Yu-Chun Kuo; Chien-Kai Wang, PhD;
Shih-He Siao; Louis W. Chang, Ph.D.; Pinpin Lin, Ph.D.
Abstract: Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen, TCDD only induced oxidative DNA damages. In our present study, we combined TCDD with 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to investigate their tumorigenic effects on lung tumor formation in A/J mice. Application of NNK at a tumorigenic dose (2 mg/mouse) induced lung adenoma in both male and female A/J mice. Neither application of NNK at a non-tumorigenic dose (1 mg/mouse) nor repeated application of TCDD alone increased tumor incidence. Following the single injection of NNK at a non-tumorigenic dose (1 mg/mouse), repeated application of TCDD significantly increased the lung tumor incidence in female, but not in male, A/J mice 24 weeks later. Utilizing the real-time RT-PCR array, we found that P16 mRNA was significantly reduced in female lung, but not male lung, of NNK/TCDD co-treated A/J mice. With immunohistochemical staining, we confirmed that nuclear P16 protein was reduced in the lungs of NNK/TCDD co-treated female mice. These data suggest that P16 reduction at least partially contributed to synergistic effects of TCDD in lung tumorigenesis.
Dear Editor:
Kindly consider the enclosed manuscript entitled “Synergism between 2,3,7,8- tetrachlorodibenzo-p-dioxin and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumor incidence in mice”, by Wang et al., for publication in Journal of
Hazardous Materials. The data presented in this manuscript have not been submitted for publication elsewhere. All authors are aware of and agree to the content of the paper and their being listed as an author on the paper. This manuscript contains 3438 words including text, figure and table legends.
Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen, its function in lung carcinogenesis is still uncertain. In our present study, we demonstrated a synergistic effect between TCDD and NNK (a tobacco-specific nitrosamine) on lung adenoma formation in female A/J mice. We further identified that TCDD reduced p16 expression in the lung of A/J mice. Our data suggest that p16 reduction at least partially contributed to the synergistic effects between TCDD and NNK in lung tumorigenesis.
All related correspondence should be sent directly to me. My mailing address, telephone number, fax number and e-mail address are listed below.
Your kind assistance in evaluating this manuscript is greatly appreciated.
Sincerely yours,
Pinpin Lin, Ph.D.
Investigator
National Health Research Institutes
Division of Environmental Health and Occupational Medicine Zhunan, Taiwan
Phone: 886-37-246166 ext 36508 Fax: 886-37-587406
E-mail: [email protected]
Cover Letter
Dear Editor:
Journal of Hazardous Materials is aimed to characterize the harmful effects of hazardous materials. Our present data characterized a synergistic harmful effect of two environmental pollutants, cigarette smoking and dioxin. The information for carcinogenic interaction of environmental pollutants in animal models is rare, especially in a lung tumor model. By publishing our data in this journal, more
scientists will be aware of the importance of chemical-chemical interaction. Our data also offer new directions in understanding environmental factors-associated lung cancer.
Sincerely yours,
Pinpin Lin, Ph.D.
Investigator
National Health Research Institutes
Division of Environmental Health and Occupational Medicine Zhunan, Taiwan
Phone: 886-37-246166 ext 36508 Fax: 886-37-587406
E-mail: [email protected]
*Novelty Statement
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Synergism between 2,3,7,8- tetrachlorodibenzo-p-dioxin and
1
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumor
2
incidence in mice
3
Ying-Jan Wanga, Han Changb, Yu-Chun Kuoc, Chien-Kai Wangc, Shih-He Siaoa, 4
Louis W. Changc, and Pinpin Linc*
5 6
a Department of Environmental and Occupational Health, National Cheng Kung 7
University, Medical College, Tainan, Taiwan 8
b Department of Pathology, School of Medicine, Chung Shan Medical University, 9
Taichung, Taiwan 10
c Division of Environmental Health and Occupational Medicine, National Health 11
Research Institutes, Zhunan, Taiwan 12
13 14 15
Correspondence: Pinpin Lin, Ph.D. Division of Environmental Health and Occupational 16
Medicine, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town, 17
Miaoli County 350, Taiwan, ROC.
18 19
Tel: 886-37246166; Fax: 886-37-587406; e-mail: [email protected] 20
21
*Manuscript
Click here to view linked References
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Abstract 22
Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human 23
carcinogen, TCDD only induced oxidative DNA damages. In our present study, we 24
combined TCDD with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to 25
investigate their tumorigenic effects on lung tumor formation in A/J mice. Application 26
of NNK at a tumorigenic dose (2 mg/mouse) induced lung adenoma in both male and 27
female A/J mice. Neither application of NNK at a non-tumorigenic dose (1 mg/mouse) 28
nor repeated application of TCDD alone increased tumor incidence. Following the 29
single injection of NNK at a non-tumorigenic dose (1 mg/mouse), repeated 30
application of TCDD significantly increased the lung tumor incidence in female, but 31
not in male, A/J mice 24 weeks later. Utilizing the real-time RT-PCR array, we found 32
that P16 mRNA was significantly reduced in female lung, but not male lung, of 33
NNK/TCDD co-treated A/J mice. With immunohistochemical staining, we confirmed 34
that nuclear P16 protein was reduced in the lungs of NNK/TCDD co-treated female 35
mice. These data suggest that P16 reduction at least partially contributed to synergistic 36
effects of TCDD in lung tumorigenesis.
37 38
Keyword: 2,3,7,8- tetrachlorodibenzo-p-dioxin, P16, lung cancer 39
40 41
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Introduction 42
The health impact of exposure to persistent organic pollutants, such as dioxins, is of 43
great concern to the general public. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is 44
the most potent dioxin congener. Epidemiological studies show that exposure to 45
TCDD increases all cancer mortality, including lung cancer [1, 2]. Long-term 46
treatment (2 years) of TCDD leads to the development of tumors of the liver, thyroid, 47
lung, and other sites in female rats [3]. In experimental studies, TCDD activates aryl 48
hydrocarbon receptors (AhRs), which change many gene expressions and possibly 49
affect cancer development [4]. Based on epidemiological data and mechanistic studies, 50
International Agency for Research on Cancer (IARC) has classified TCDD as a 51
human carcinogen since 1997. However, Cole et al. [5] indicated that the increase in 52
human cancer risk was only modest when people were exposed to TCDD. They 53
believed that cancer risk increased when TCDD exposure was combined with other 54
environmental factors, such as cigarette smoking.
55
Unlike to most carcinogens, TCDD does not directly produce DNA adducts or DNA 56
damage. Nonetheless, TCDD increases oxidative stress [6, 7]. Oxidative stress is one 57
of the mechanisms of tumor promotion [8, 9]. Currently, there are only three studies 58
that investigated the promotion effects of TCDD in lung tumors, and one of them 59
reported positive results [10, 11]. While both single treatment with 60
N-nitrosodimethylamine (NDMA) and the combined treatment of NDMA plus TCDD 61
induced lung tumors in 100% of animals, the multiplicity of lung tumors was 62
increased in the lungs of NDMA/TCDD co-treated mice [11]. The other two studies 63
utilized diethyl-N-nitrosamine (DEN) as the tumor initiator, which failed to show the 64
growth promotion effect of TCDD for lung tumors in either mice or rats [10]. It 65
appears that TCDD did not universally promote lung tumorigenicity, but varied 66
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depending on the kind of tumor initiator used. Furthermore, neither DEN nor NDMA 67
is present in the environment, the synergistic effect between TCDD and other 68
chemicals to which humans are also exposed should be investigated [12]. Cigarette 69
smoke is one of the major environmental risk factors for lung cancer development. A 70
tobacco-specific N-nitrosamine, 4-(methylnitrosamino) -1-(3-pyridyl)-1-butanone 71
(NNK) plays an important role in tobacco-related human lung cancer [13].
72
Furthermore, NNK induces lung adenoma/adenocarcinoma in A/J mice [14], which is 73
often used as an animal model for lung carcinogenesis studies. Therefore, in our 74
present study we evaluated the synergistic effects between TCDD and NNK in A/J 75
mice.
76
Results generated from mechanistic studies are one of the reasons why TCDD is 77
classified as a human carcinogen. For example, TCDD induces matrix 78
metalloproteinase expression and invasion in melanoma cells [15]. TCDD modulated 79
cell plasticity and mobility in a Jun NH2-terminal kinase dependent mechanism [16].
80
Ray and Swanson [17] reported that TCDD inhibited culture-induced senescence in 81
keratinocytes. However, most (or all) of these data were obtained in vitro. In our 82
present study, we planned to identify cancer-related genes modulated by TCDD in 83
vivo.
84 85
Materials and Methods 86
Animals 87
A/J mice (6 weeks of age) , acquired from the animal center of the National Cheng 88
Kung University Medical College, were housed five per cage at 24 ± 2°C and 50% ± 89
10% relative humidity and subjected to a 12-h light/12-h dark cycle. They were 90
acclimatizedfor 1 week before use and fed with a Purina chow diet and water ad 91
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libitum.
92 93
Female mice were randomly divided into five groups (groups I to V). Group I (n = 37) 94
were given a single injection of 0.1 ml normal saline (vehicle) intraperitoneally per 95
mouse as the negative control. Group II (n = 19) were given a single high dose of 96
NNK (2 mg/0.1 ml saline/mouse intraperitoneally) as the positive control. Group III 97
(n = 36) were given a single low dose of NNK (1 mg/0.1 ml saline/mouse 98
intraperitoneally). Group IV (n = 22) were given a loading dose of 5 μg of TCDD/kg 99
of body weight, followed by weekly maintenance doses of 1.42 μg of TCDD/kg of 100
body weight administered intraperitoneally. Group V (n = 36) were given a low dose 101
of NNK for 1 week, then a loading dose of 5 μg of TCDD/kg of body weight, 102
followed by weekly maintenance doses of 1.42 μg of TCDD/kg of body weight 103
administered intraperitoneally. The experiments were terminated 24 weeks after the 104
first treatment. Male mice were randomly divided into four groups (groups I to IV), 105
including vehicle control (n = 20), high dose of NNK (2 mg/mouse, n = 7), low dose 106
of NNK (1 mg/mouse, n = 20) and low dose NNK plus TCDD (n = 19). Figure 1 107
showed the full Schedule for animal treatments.
108 109
All of the surviving mice were sacrificed under ether anesthesia. At autopsy, their 110
lungs were excised and weighed, infused with 10% neutral buffered formalin, and 111
inspected grossly. All of the lung tumors were macroscopically observed, and 112
tumor-bearing lung lobes were examined histopathologically.
113 114
Histopathology 115
Lung tissues were sliced and immediately fixed in 10% neutral buffered formalin for 116
16–48 hours before being processed with standard histopathological tissue methods, 117
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including ethanol dehydration and paraffin embedding. All lung tissues were sliced 118
into 5-m thick sections. Lung sections were then deparaffinized in xylene and 119
rehydrated through graded ethanol solutions to distilled water. Initially, one section 120
was stained with hematoxylin and eosin (H-E) for examination of the lung lesions 121
including hyperplasia, adenoma, and malignancy diagnosed according to the criteria 122
of Tumors of the Mouse [18]. When the inconsistent findings between macroscopic 123
and microscopic examinations were observed, 10 serial sections were then cut and 124
number-labeled. The odd numbered sections were stained with H-E for further 125
confirmation of the presence of tumor formation and the enumeration of the lung 126
tumors microscopically. The mouse without any tumor was defined as a negative 127
tumor-bearing mouse. The rest of sections were subjected to performance of 128
immunohistochemical assays for P16 protein.
129 130
Analysis of the real-time reverse transcription polymerase chain reaction 131
(RT-PCR) array 132
Total RNA was prepared using TriReagent (Life Technologies, Rockville, MD, USA) 133
and the phenol-chloroform extraction method. Synthesis of cDNA was performed 134
using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase, 135
deoxynucleotides, and RNase inhibitor (Promega, Madison, WI, USA), with 2 μg of 136
total RNA mixed with 250 ng of random primer (BioLabs, Beverly, MA, USA). Eight 137
representative lung cDNA samples were selected from among the vehicle, NNK, 138
TCDD, and NNK plus TCDD groups. Between groups, the relative gene expression 139
was measured using the mouse Cancer PathwayFinder (PAMM-033, Superarray, 140
Frederick, MD), which includes 84 genes involved in transformation and 141
tumorigenesis.
142 143
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Real-time RT-PCR assay 144
Quantitative PCR of cyclin-dependent kinase inhibitor 2A (Cdkn2a, P16) and 145
glyceraldehyde-3-phosphate dehydrogenase (GAPHD) were performed using the 146
TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems, Foster City, 147
CA) and the ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied 148
Biosystems). The P16 primers and probes were the Assay-on-Demand Gene 149
Expression Assay Mix (Perkin-Elmer Applied Biosystems). Each data point was 150
repeated four times. Quantitative values were obtained from the threshold cycle (CT) 151
number. The relative mRNA levels of P16 = 2-ΔCt, ΔCt= CtP16-CtGAPDH. 152
153
P16 immunohistochemistry 154
In order to confirm the expression and location of P16 protein in the lung tissues, 155
immunohistochemistry was performed as previously described [19]. The specific 156
primary antibody for P16 (1:400 dilution; Clone F-12, Santa Cruz Biotechnology, 157
Santa Cruz, CA), the Universal LSAB2 kit (DakoCytomation, Glostrup, Denmark), 158
and the Chromogen DAB+ system (DakoCytomation) were used for detecting the 159
immunoreactivity. The step of antigen retrieval was not essential for P16 antibody.
160
The P16 staining was reactive to the nucleus and cytoplasm of lung tumor cells and 161
respiratory epithelial cells. P16 was occasionally expressed in the nuclei of stroma 162
cells in the lung sections, while the interstitial stroma was always P16-negative. When 163
the staining intensity of nucleus and/or cytoplasm in stained cells was stronger than 164
that of the stroma, the cells were defined as P16 nuclear and/or cytoplasmic positive 165
cells. Furthermore, lung tumors having more than 10% P16-nuclear positive cells 166
were regarded as P16-positive tumors. The human tonsil collected from Chung Shan 167
Medical University Hospital was used as a positive control [20]. When primary 168
antibody was replaced with normal serum and phosphate buffer in the procedure, the 169
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results were used as negative controls.
170 171
Statistics 172
Student’s t-test was used to compare the results of anchorage-independent growth 173
assays and real-time RT-PCR assay among the groups. Fisher’s exact test was used to 174
compare the incidence of tumor formation between treated and control groups. The 175
two-sided P values less than 0.05 were considered significant.
176 177
Results 178
Effects of TCDD and NNK on lung adenoma formation 179
Gross examination of almost all lung tumors showed that they were located in the 180
subpleural areas. They were well-circumscribed, white in color, and ranged in size 181
from 1 to 3 mm3 at their greatest dimension. Microscopically, all of the lung tumors 182
featured histology consistent with adenoma. No other pathological lesions including 183
hyperplasia, dysplasia, or adenocarcinoma were identified in the sections examined.
184
Tumor incidences for each treated and control group is listed in Table 1. The gender 185
difference in TCDD-promoted lung tumorigenesis was verified in A/J mice. Both 186
female and male A/J mice had 8% and 10% of spontaneous lung adenomas, 187
respectively (Table 1). While treatment with high-dose NNK (2 mg/mouse) 188
significantly increased tumor incidences in both genders (84% in females and 71% in 189
males, both P = 0.001 and 0.005 respectively), treatment with low-dose NNK (1 190
mg/mouse) only slightly increased tumor incidences (19% in females and 15% in 191
males, P = 0.190 and 1.000 respectively. However, combined treatment with low-dose 192
NNK and TCDD significantly increased the incidence to 36% in females, but 193
increased the incidence to 26% in males without significance (Table 1). Treatment 194
with TCDD resulted in a low incidence (9%) of adenomas in females. It appears that 195
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the synergistic effect between TCDD and NNK was significant in females, but not in 196
males.
197 198
Effects of TCDD on p16 expression in lungs 199
To understand the mechanisms of the tumor growth promotional effect of TCDD, we 200
screened for the differential expression of 84 cancer-related genes in female mouse 201
lungs using a real-time RT-PCR array. Gene expression modulated by treatment with 202
low-dose NNK (LNNK), TCDD, or LNNK/TCDD is shown in Table 1. Expression of 203
12, 10, or 9 cancer-related genes was modulated in LNNK, TCDD, or 204
LNNK/TCDD-treated groups, respectively. P16 is a tumor suppressor gene and its 205
expression is commonly reduced in human lung cancer specimens (Belinsky, 2004).
206
In our experiments, P16 was modulated in the LNNK-treated group and was uniquely 207
down regulated in the LNNK/TCDD-treated group (Table 2). Furthermore, the 208
reduction of P16 mRNA was only observed in female, but not in male, mice 209
co-treated with NNK/TCDD (Figure 2).
210
The reduction of P16 protein levels was further confirmed by immunostaining. Most 211
bronchiolar and alveolar cells displayed positive P16 immunostaining in the nuclei 212
and cytoplasm of control mice (Figure 3, A and B). However, the results of 213
immunostaining in lung tumor cells of NNK/TCDD co-treated groups were different 214
in male and female mice. In the female NNK/TCDD co-treated group, P16 nuclear 215
immunostaining occurred in 43% of lung tumors in which a weak P16 staining was 216
observed in nuclei, cytoplasm or both (Figure 3C). In contrast, in the male 217
NNK/TCDD co-treated group, P16 nuclear immunostaining occurred in 75% of lung 218
tumors in which tumor cells showed a moderately cytoplasmic or strongly P16 219
nuclear staining (Figure 3D). It appears that TCDD reduced P16 expression in the 220
lungs of A/J mice in a gender-dependent manner.
221
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222
Discussion 223
Cigarette smoke and dioxin are classified as human carcinogens. NNK is the potent 224
carcinogen in cigarette smoke and TCDD is the most potent AhR agonist among 225
dioxins. Because NNK metabolites and TCDD are detectable in human specimens of 226
the general population [21-24], it is possible that NNK may interact with TCDD and 227
enhance cancer risks in the general population, such as lung cancer. In our present 228
study, we demonstrated that the combined treatment of NNK and TCDD at 229
non-carcinogenic doses significantly increased the incidence of lung adenoma in A/J 230
mice. Furthermore, this carcinogenic effect was more common in female than in male 231
mice.
232 233
Although low-dose NNK (1 mg/mouse) or TCDD alone failed to increase the 234
incidence of lung adenoma in female mice, low-dose NNK or TCDD modulated 235
expression of several cancer-related genes. Low-dose NNK reduced expression of 236
nine genes and four of them (Mta2, Mmp9, S100A4, and Plau) involved invasion and 237
metastasis. It is consistent with the phenomenon that NNK tends to induce adenoma 238
in mice, which is a non-invasive phenotype. TCDD alone increased expression of nine 239
genes with diverse functions, but only transformation related protein 53 (P53) was 240
also induced in LNNK/TCDD co-treated mice. On the other hand, P16 expression 241
was only reduced in LNNK/TCDD co-treated mice. It appears that a synergistic 242
interaction occurred between low dose NNK and TCDD. This interaction not only 243
reduced P16 expression, but also increased the tumor incidence in female mice.
244 245
Cellular senescence is proposed to be a tumor-suppressive mechanism that stops 246
incipient cancer cells from proliferating. The hallmark of cellular senescence is an 247
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inability to progress through the cell cycle. The P16- retinoblastoma protein (pRB) 248
tumor suppressor pathway is one of the pathways that controls senescent growth arrest 249
[25]. Some senescence-inducing stimuli, such as oncogenes and DNA-damage 250
responses, activate the P16-pRB and P53 pathways. Previously, Ray and Swanson [26]
251
demonstrated that TCDD attenuated senescence and repressed expression of P16 as 252
well as P53 in primary human keratinocytes. In the present study, mRNA and protein 253
levels of P16 were significantly reduced in the lungs of NNK/TCDD co-treated 254
female mice. Furthermore, P16 reduction correlated with tumor incidence in a gender 255
dependent manner. These results imply that P16 reduction might involve in the 256
synergisitc effect of TCDD in the lungs of female mice. These data are also consistent 257
with results reported by other studies that loss of P16 function in mice increased 258
susceptibility to carcinogens [27, 28].
259 260
Loss of P16 expression occurs in 30%–70% of human non-small cell lung cancers, 261
and is more prevalent in smokers than in nonsmokers [29]. In human lung cancer, loss 262
of P16 expression typically results from allelic loss in combination with 263
hypermethylation of the P16 promoter [30-33]. Some studies also reported that 264
histone modifications regulated P16 expression [34-36]. Belinsky et al. [37] reported 265
that NNK treatment reduced P16 expression in approximately half of lung 266
tumor-bearing A/J mice. They further suggested that reduced P16 expression in some 267
of these tumors was attributed to deletion of the p16 gene, but not hypermethylation 268
of P16 promoter [37, 38]. On the other hand, Ray and Swanson et al. [26] reported 269
that TCDD induced methylation of the P16 promoter in primary human keratinocytes.
270
In our present study, we observed reduced P16 expression in NNK/TCDD co-treated 271
female mice. The levels of P16 protein were reduced in both the nuclei and cytoplasm 272
of the tumor cells of NNK/TCDD co-treated female mice. The most striking result 273
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was that the P16 reduction by NNK/TCDD in the lung was gender specific. DNA 274
methyltransferases catalyze DNA methylation. We examined the expression of DNA 275
methyltransferase 1 (DNMT1) protein in these specimens, but DNMT1 expression did 276
not correlate with the loss of P16 protein in these specimens (data not shown). The 277
mechanism remains to be clarified in the future.
278 279
Acknowledgements 280
This study was supported by a grant (EO-097-PP-03) from the Division of 281
Environmental Health and Occupation Medicine, National Health Research Institutes 282
Taiwan. The scientific content of this manuscript does not necessarily signify the 283
views and policies of the DEHOM/NHRI or condemn, endorse, or recommend for use 284
anything presented in this article.
285
Declarations of interest 286
The Author(s) declare that they have no competing interests.
287
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Table 1. The incidence of lung tumor formation in A/J mice treated with NNK and/or 418
TCDD.
419
Gender Treatment Mice numbers per group
Number (%) of tumor-bearing mice
Female Control 37 3 (8)
HNNK 19 16 (84)*
LNNK 36 7 (19)
TCDD 22 2 (9)
LNNK + TCDD 36 13 (36)*
Male Control 20 2 (10)
HNNK 7 5 (71)*
LNNK 20 3 (15)
LNNK + TCDD 19 5 (26)
Control, injected with sterile normal saline; low dose of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (LNNK), injected with 1 mg NNK/mouse; high dose of NNK (HNNK), injected with 2 mg NNK/mouse; 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), injected with 5μg/kg TCDD once and then 1.42 μg/kg TCDD for three times weekly.
*, P < 0.05, compared with control group by using two tailed Fisher’s exact test.
420 421 422 423 424 425 426 427 428 429 430
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Table 2. Gene expression modulated by NNK and/or TCDD in the lung of female 431
mice.
432
Treatment Symbol Description Fold
LNNK Twist1 Twist gene homolog 1 (Drosophila) 3.43
Brca1 Breast cancer 1 1.5
Casp8 Caspase-8/FLICE 1.49
Mta2 Metastasis-associated gene family, member 2 0.67 Plau Plasminogen activator, urokinase 0.67 Birc5 Baculoviral IAP repeat-containing 5 0.56
Jun Jun oncogene 0.53
S100a4 S100 calcium binding protein A4 0.53 Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21) 0.51
Fos FBJ osteosarcoma oncogene 0.45
Mcam Melanoma cell adhesion molecule 0.4
Mmp9 Matrix metallopeptidase 9 0.18
TCDD Twist1 Twist gene homolog 1 (Drosophila) 4.09 Ncam1 Neural cell adhesion molecule 1 2.37 Tnfrsf10b Tumor necrosis factor receptor superfamily,
member 10b
1.93
Casp8 Caspase-8/FLICE 1.82
Trp53 Transformation related protein 53 1.82 Egfr Epidermal growth factor receptor 1.76
Myc Myelocytomatosis oncogene 1.55
Nme4 Non-metastatic cells 4, protein expressed in 1.51
Hgf Hepatocyte growth factor 1.46
Kiss1 KiSS-1 metastasis-suppressor 0.46 LNNK+TCD
D
Brca1 Breast cancer 1 1.75
Trp53 Transformation related protein 53 1.6 Plau Plasminogen activator, urokinase 0.67 Birc5 Baculoviral IAP repeat-containing 5 0.64
Jun Jun oncogene 0.57
Cdkn2a Cyclin-dependent kinase inhibitor 2A 0.56
Fos FBJ osteosarcoma oncogene 0.46
S100a4 S100 calcium binding protein A4 0.45
Mmp9 Matrix metallopeptidase 9 0.11
Each group contained eight animals. Data of low dose of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (1 mg/0.1 ml saline/mouse intraperitoneally, LNNK), 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) or NNK/TCDD-treated group were compared with those of control group. The fold changes of list genes were statistical significant (p < 0.05).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Figure legends 433
Figure 1. The schedule for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone 434
(NNK) and/or
2,3,7,8-
tetrachlorodibenzo-p-dioxin(
TCDD) treatment in A/J 435mice. The control was intraperitoneally injected with phosphate-buffered saline on the 436
first day. The HNNK group was intraperitoneally injected with 2 mg of NNK per 437
mouse on the first day. The LNNK group was intraperitoneally injected with 1 mg of 438
NNK per mouse on the first day. The TCDD was intraperitoneally injected with 5 439
μg/kg of TCDD once for the first week, followed by weekly injections of 1.42 μg/kg 440
of TCDD for 3 weeks.
441
442
Figure 2. Effects of NNK/TCDD co-treatment on p16 mRNA levels in male and 443
female A/J mice. P16 mRNA levels were determined with the real-time RT-PCR 444
method. Each group contained eight animals, and each data point was repeated for 445
four times. *p < 0.05, compared to the female control group.
446
447
Figure 3. Immunohistochemical staining of P16 in control and 448
LNNK/TCDD-treated lung of mice. A and B. In female and male mice of control 449
groups, many alveolar and airway epithelial cells demonstrated strong staining of P16 450
in the cell nucleus and/or cytoplasm. C. In LNNK/TCDD treated female mice, some 451
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
tumor cells demonstrated P16 staining in the cell cytoplasm and/or nucleus. D. In 452
LNNK/TCDD-treated male mice, many tumor cells demonstrated P16 positive 453
staining in both the cell nucleus and cytoplasm. Scale bar, 50 μm.
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Figure 1
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Figure 2
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Figure 3
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Responses to technique check:
Comments:
1) Cover letter provided should state the manuscript word count, which includes text, figures, and table legends, but not references.
Ans. The word count for this manuscript is 3438 words, and stated in the attached cover letter now.
2) The novelty statement (100 words maximum) provided should also explain: why the work should be published in the Journal of Hazardous Materials.
Ans. A rewritten novelty statement is now attached for explaining the reason to publish this manuscript in the Journal of Hazardous materials.
3) Reference list must conform strictly to the guide for authors. For journal articles, please adhere to the following order: initials followed by surname, article title, abbreviated journal name, volume/issue number, year in parenthesis and page span.
Ans. The reference list is now rearranged by Endnote X following the order given by Journal of Hazardous materials.
4) The figures should be cited sequentially in the text. Figure 1 provided in the
manuscript is not cited in the text and figure 4 cited in the manuscript is not provided.
Ans. Figure 1 is now stated on the first paragraph of materials and methods section.
Figure 4 actually indicated figure 3, and already corrected now. Many thanks for reminding this error.
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